Injectable and moldable bone substitute materials

ABSTRACT

An osteoimplant composite comprising a plurality of particles of an inorganic material, a bone substitute material, a bone-derived material, or any combination thereof; and a polymer material with which the particles are combined. The composite is either naturally moldable or flowable, or it can be made moldable or settable. After implantation, the composite may be set to provide mechanical strength to the implant. The inventive composite have the advantage of being able to fill irregularly shape implantation site while at the same time being settable to provide the mechanical strength required for most orthopedic applications. The invention also provides methods of using and preparing the moldable and flowable composites.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §120 and is acontinuation of U.S. patent application, U.S. Ser. No. 11/625,119, filedJan. 19, 2007, which is a continuation-in-part of U.S. patentapplication, U.S. Ser. No. 10/735,135, filed Dec. 12, 2003, which claimspriority under 35 U.S.C. §119(e) to provisional patent application, U.S.Ser. No. 60/432,968, filed Dec. 12, 2002, now expired; each of which isincorporated herein by reference. The present application also claimspriority under 35 U.S.C. §120 and is a continuation-in-part of U.S.patent application, U.S. Ser. No. 11/047,992, filed Jan. 31, 2005, whichclaims priority under 35 U.S.C. §119(e) to provisional patentapplication, U.S. Ser. No. 60/568,472, filed May 4, 2004; each of whichis incorporated herein by reference. The present application also claimspriority under 35 U.S.C. §119(e) to U.S. provisional patentapplications, U.S. Ser. Nos. 60/760,538, 60/760,752, 60/760,753, and60/760,239, all of which were filed Jan. 19, 2006, each of which isincorporated herein by reference.

FIELD OF THE INVENTION

This invention pertains to injectable and moldable polymer-bonecomposites that set upon exposure to certain predetermined conditionsfor use in orthopedic medicine.

BACKGROUND OF THE INVENTION

Bone is a composite material composed of impure hydroxyapatite,collagen, and a variety of non-collagenous proteins, as well as embeddedand adherent cells. Bone can be processed into an implantablebiomaterial, such as an allograft, for example, by removing the cells,leaving behind the extracellular matrix. The processed bone biomaterialcan have a variety of properties, depending upon the specific processesand treatments applied to it, and may incorporate characteristics ofother biomaterials with which it is combined. For example, bone-derivedbiomaterials may be processed into load-bearing mineralized grafts thatsupport and integrate with the patient's own bone or may alternativelybe processed into soft, moldable, or flowable demineralized bonebiomaterials that have the ability to induce a cellular healingresponse.

The use of bone grafts and bone substitute materials in orthopedicmedicine is well known. While bone wounds can regenerate without theformation of scar tissue, fractures and other orthopedic injuries take along time to heal, during which the injured bone is unable to supportphysiologic loading. Metal pins, screws, and meshes are frequentlyneeded to replace the mechanical functions of injured bone. However,metal is significantly stiffer than bone. Use of metal implants mayresult in decreased bone density around the implant site due to stressshielding. Furthermore, most metal implants are permanent and unable toparticipate in physiological remodeling.

Bone's cellular healing processes, through bone tissue formation byosteoblast cells coordinated with bone and graft resorption byosteoclast cells, permit bone grafts and certain bone substitutematerials to remodel into endogenous bone that is almostindistinguishable from the original. However, the use of bone grafts islimited by the available shape and size of grafts and the desire tooptimize both mechanical strength and degradation rate. Variations inbone size and shape among patients (and donors) also make bone grafts aless optimal substitute material. Bone substitute materials and bonechips are quickly remodeled but cannot immediately provide mechanicalsupport. In contrast, cortical bone grafts can support physiologicalstresses but remodel slowly.

U.S. Pat. Nos. 5,899,939; 5,507,813; 6,123,731; 6,294,041; 6,294,187;6,332,779; 6,440,444; and 6,478,825; the contents of all of which areincorporated herein by reference, describe methods for preparingcomposites including allogenic bone for use in load bearing orthopedicapplications.

Thus, it is desirable to have a bone substitute material for structuralgrafts that may be produced in larger quantities than grafts derivedsolely from bone and that may be fabricated or molded into shapeswithout being limited by the shape of the originating tissue. It is alsodesirable to have injectable bone substitute materials that may beimplanted using minimally invasive techniques.

SUMMARY OF THE INVENTION

The present invention stems from the recognition that bone substitutematerial is needed that is moldable or injectable to fill irregularlyshaped volumes in or near a bone. A bone substitute material that ismoldable and/or flowable while being implanted but later becoming setwith a desired degree of mechanical strength would be particularlyuseful in treating bony defects in a subject. The bone substitutematerial could be molded, shaped, or injected into the site ofimplantation and then set under predetermined suitable conditions suchas cooling to body temperature. The set material would provide thedesired mechanical strength for the implantation site reducing the needfor metal pins, screws, or meshes. The present invention provides suchbone substitute composite materials made up of particles of inorganicmaterial, a bone substitute material, and/or a bone-derived material,and a polymer, wherein the composite is moldable or flowable, and it canbe set upon exposure to suitable conditions. Processes for preparing andusing these materials, and kits for easy administration of the inventivematerials are also provided.

In one aspect, the invention provides compositions including a pluralityof particles of an inorganic material, a bone substitute material, abone-derived material, or any combination thereof, and a polymer withwhich the particles are combined. The composite of the particles and thepolymer is naturally moldable or flowable, or the composite can be mademoldable or flowable such as by heating or by the addition of a solvent.The composition may range from a thick, flowable liquid to a moldable,dough-like substance. In certain embodiments, the composite has a lowenough viscosity to be suitable for injection. In other embodiments, thecomposite is workable so that it can be molded into an implantationsite. The composite becomes set upon exposure to certain predeterminedsuitable conditions. The conditions for setting will of course depend onthe composite being used. Exemplary conditions for setting the compositemay include a change in temperature (e.g., heating or cooling), a changein osmotic pressure, exposure to electromagnetic radiation (e.g.,microwaves, IR radiation, visible light, UV radiation), cross-linkingthe composite, exposing the composite to a chemical agent, a change inthe content of water or other solvent in the composite, a change in thecontent of a component of the composite, or a change in a diffusiongradient. The particles in the composite have an average size of about10 to about 1000 microns in diameter, preferably an average size ofabout 20 to about 800 microns in diameter. In certain embodiments, themedian size of the particles ranges from about 10 to about 1000 micronsin diameter, preferably from about 20 to about 800 microns. Smaller orlarge particles may also be found in the composite. A particle sizedistribution of the particles with respect to a median value may be plusand minus about 90% or less, about 50% or less, or about 20% or less. Incertain embodiments, at least about 60% of the particles have a mediansize of about 10 microns to about 1000 microns in their greatestdimension. In certain embodiments, at least about 60% of the particleshave a median size of about 20 microns to about 800 microns in theirgreatest dimension.

The polymer used in preparing the inventive composite may be selectedfrom monomers, pre-polymers, oligomers, polymers, cross-linked polymers,partially polymerized polymers, partially cross-linked polymers, and anycombinations thereof. For example, the composite may include monomers,oligomers, and polymers. Exemplary polymers useful in the inventivecomposites include, but are not limited to, poly(lactide),poly(glycolide), poly(lactide-co-glycolide),poly(D,L-lactide-co-glycolide), poly(L-lactide-co-glycolide),poly(caprolactone), polyurethane, polycarbonates, polyarylates,polypropylene fumarates), polyphosphazines, and combinations, blends, orco-polymers thereof.

In certain embodiments, the composite include particles of bone-derivedmaterial. The bone-derived material of such composites may include oneor more of nondemineralized bone particles, demineralized boneparticles, lightly demineralized bone particles, and deorganified boneparticles. The bone-derived material may include one or more of corticalbone, cancellous bone, and cortico-cancellous bone. Also, thebone-derived material may include autogenous bone, allogenic bone, andxenogeneic bone. In certain embodiments, the composite includes aninorganic material (e.g., an inorganic ceramic) and/or a bone substitutematerial. Exemplary inorganic materials or bone substitute materialsuseful in the inventive composites include aragonite, dahlite, calcite,amorphous calcium carbonate, vaterite, weddellite, whewellite, struvite,urate, ferrihydrite, francolite, monohydrocalcite, magnetite, goethite,dentin, calcium carbonate, calcium sulfate, calcium phosphosilicate,sodium phosphate, calcium aluminate, calcium phosphate, hydroxyapatite,α-tricalcium phosphate, dicalcium phosphate, β-tricalcium phosphate,tetracalcium phosphate, amorphous calcium phosphate, octacalciumphosphate, BIOGLASS™, fluorapatite, chlorapatite, magnesium-substitutedtricalcium phosphate, carbonate hydroxyapatite, substituted forms ofhydroxyapatite (e.g., hydroxyapatite derived from bone may besubstituted with other ions such as fluoride, chloride, magnesium,sodium, potassium, etc.), and combinations and derivatives thereof. Incertain embodiments, the particles themselves are composites thatinclude one or more of an inorganic material, a bone substitutematerial, and a bone-derived material; and one or more of bovine serumalbumin, collagen, an extracellular matrix component, a syntheticpolymer, and a natural polymer. The composite may range fromapproximately 10% particles to about 95% particles by weight, forexample, approximately 50% particles to approximately 80% particles byweight. In certain embodiments, the composite is approximately 50%,approximately 55%, approximately 60%, or approximately 65% particles byweight. The composite may also include other components. For example,the composite may further include one or more of an initiator,accelerator, catalyst, solvent, wetting agent, lubricating agent,labeling agent, plasticizer, radiopacifier, porogen, bioactive agent,biostatic agent, cell, polynucleotide, protein (e.g., bone morphogenicprotein, cytokine, growth factor, aniogenic factor), pharmaceuticalagent (e.g., anti-inflammatory agent, analgesic, antibiotic, etc.), andpharmaceutically acceptable excipient. In certain embodiments, thecomposite includes a plasticizer that softens the composite making itmore pliable. Exemplary plasticizer include glycerol and poly(ethyleneglycol) (PEG) (e.g., PEG 8000, PEG 6000, PEG 4000). In certainembodiments, the polymer component of the composite includes PEGblended, grafted, or co-polymerized with the polymer. In certainembodiments, the composite includes a porogen that diffuses, dissolves,and/or degrades after implantation of the composite leaving a pore. Theporogen may be a gas (e.g., carbon dioxide, nitrogen), liquid (e.g.,water), or solid (e.g., crystalline salt). The porogen may be awater-soluble chemical compound such as a carbohydrate (e.g.,poly(dextrose), dextran), salt, polymer (e.g., polyvinyl pyrrolidone),protein (e.g., gelatin), pharmaceutical agent (e.g., antibiotics), smallmolecule, etc.

In another aspect, the invention provides a method of administering aninventive composite to a subject in need thereof. The inventivecomposites are particularly useful in orthopedic medicine. The compositemay be used to repair a fracture or other bony defect in a subject'sbone. The method includes providing a flowable or moldable composite ofa polymer and a plurality of particles including one or more of aninorganic material, a bone substitute material, and a bone-derivedmaterial; administering the composite to a subject in need thereof; andcausing the composite to set. Before administration, the composite maybe made flowable or moldable by heating the composite or adding asolvent to the composite. The composite is administered into animplantation site (e.g., a bony defect) followed by setting thecomposite. The composite may be set by allowing the composite to come tobody temperature, increasing the molecular weight of the polymer in thecomposite, cross-linking the polymer in the composite, irradiating thecomposite with UV radiation, adding a chemical agent to the polymer, orallowing a solvent to diffuse from the composite. The set osteoimplantcomposite is allowed to remain at the site providing the strengthdesired while at the same time promoting healing of the bone and/or bonegrowth. The polymer component of the composite may degraded or beresorbed as new bone is formed at the implantation site. The polymer maybe resorbed over approximately 1 month to approximately 6 years. Thecomposite may start to be remodeled in as little as a week as thecomposite is infiltrated with cells or new bone in-growth. Theremodeling process may continue for weeks, months, or years.

In yet another aspect, the invention provide a method of preparing theinventive composites by combining a plurality of particles comprising aninorganic material, a bone substitute material, a bone-derived material,or combinations thereof; and a polymer (e.g., polycaprolactone,poly(lactide), poly(glycolide), poly(lactide-co-glycodide),polyurethane); and heating the resulting composite until is becomesmoldable (e.g., to a temperature between approximately 40° C. andapproximately 80° C.). Once the composite is implanted and allowed tocool to body temperature (approximately 37° C.), the composite becomesset. The invention also provides another method of preparing theinventive composites by combining a plurality of particles comprising aninorganic material, a bone substitute material, a bone-derived material,or combinations thereof; and a polymer (e.g., polycaprolactone,poly(lactide), poly(glycolide), poly(lactide-co-glycodide),polyurethane); and adding a solvent or pharmaceutically acceptableexcipient so that the resulting composite is flowable or moldable. Thecomposite may then be injected or placed into the site of implantation.As the solvent or excipient diffuses out of the composite, it becomesset in place.

In another embodiment, the invention provides kits for the treatment ofbone. The kit includes a composition including a plurality of particlesincluding one or more of an inorganic material, a bone substitutematerial, and a bone-derived material; and a polymer with which theparticles are combined, the composition being contained within adelivery system for delivering the composite by injection (e.g., asyringe). The kit may also include a high pressure injection device forimplanting composite of higher viscosity. The injection device mayoperate by hydraulic or pneumatic means. The kit may also include thecomponents of the composite packaged separately for mixing just prior toimplantation. The composite is preferably sterilely packaged. In certainembodiments, the entire kit is sterilely packaged for use in a sterileenvironment such as an operating room. Various amounts of the compositemay be packaged in a kit. For larger implantation sites, kits withgreater amounts of composite are used. The amount of composite packagedin a kit may depend on the procedure being performed on the subject. Incertain embodiments, multiple individually packaged amounts of compositeare included in one kit. That way only the necessary number of packagesneed be opened for a procedure. The kit may also include a heatingapparatus for warming the composite to a temperature where it ismoldable. The kit may also include a solvent or pharmaceuticallyacceptable excipient for combining with the composite. The kit mayfurther include instructions for using the composite.

DEFINITIONS

As used herein, “bioactive agent” is used to refer to compounds orentities that alter, promote, speed, prolong, inhibit, activate, orotherwise affect biological or chemical events in a subject (e.g., ahuman). For example, bioactive agents may include, but are not limitedto osteogenic, osteoinductive, and osteoconductive agents, anti-HIVsubstances, anti-cancer substances, antibiotics, immunosuppressants,anti-viral agents, enzyme inhibitors, neurotoxins, opioids, hypnotics,anti-histamines, lubricants, tranquilizers, anti-convulsants, musclerelaxants, anti-Parkinson agents, anti-spasmodics and musclecontractants including channel blockers, miotics and anti-cholinergics,anti-glaucoma compounds, anti-parasite agents, anti-protozoal agents,and/or anti-fungal agents, modulators of cell-extracellular matrixinteractions including cell growth inhibitors and anti-adhesionmolecules, vasodilating agents, inhibitors of DNA, RNA, or proteinsynthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal andnon-steroidal anti-inflammatory agents, anti-angiogenic factors,angiogenic factors, anti-secretory factors, anticoagulants and/orantithrombotic agents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotics, targeting agents, chemotacticfactors, receptors, neurotransmitters, proteins, cell responsemodifiers, cells, peptides, polynucleotides, viruses, and vaccines. Incertain preferred embodiments, the bioactive agent is a drug. In certainembodiments, the bioactive agent is a small molecule.

A more complete listing of bioactive agents and specific drugs suitablefor use in the present invention may be found in “PharmaceuticalSubstances: Syntheses, Patents, Applications” by Axel Kleemann andJurgen Engel, Thieme Medical Publishing, 1999; the “Merck Index: AnEncyclopedia of Chemicals, Drugs, and Biologicals”, Edited by SusanBudavari et al., CRC Press, 1996, the United StatesPharmacopeia-25/National Formulary-20, published by the United StatesPharmcopeial Convention, Inc., Rockville Md., 2001, and the“Pharmazeutische Wirkstoffe”, edited by Von Keemann et al.,Stuttgart/New York, 1987, all of which are incorporated herein byreference. Drugs for human use listed by the U.S. Food and DrugAdministration (FDA) under 21 C.F.R. §§330.5, 331 through 361, and 440through 460, and drugs for veterinary use listed by the FDA under 21C.F.R. §§500 through 589, all of which are incorporated herein byreference, are also considered acceptable for use in accordance with thepresent invention.

As used herein, “biodegradable”, “bioerodable”, or “resorbable”materials are materials that degrade under physiological conditions toform a product that can be metabolized or excreted without damage to thesubject. In certain embodiments, the product is metabolized or excretedwithout permanent damage to the subject. Biodegradable materials may behydrolytically degradable, may require cellular and/or enzymatic actionto fully degrade, or both. Other degradation mechanisms, e.g., thermaldegradation due to body heat, are also envisioned. Biodegradablematerials also include materials that are broken down within cells.Degradation may occur by hydrolysis, enzymatic processes, phagocytosis,or other processes.

The term “biocompatible”, as used herein, is intended to describematerials that, upon administration in vivo, do not induce undesirableside effects. The material preferably does not induce irreversible,undesirable side effects. In certain embodiments, a material isbiocompatible if it does not induce long term undesirable side effects.In certain embodiments, the risks and benefits of administering amaterial are weighed in order to determine whether a material issufficiently biocompatible to be administered to a subject.

The term “biomolecules”, as used herein, refers to classes of molecules(e.g., proteins, amino acids, peptides, polynucleotides, nucleotides,carbohydrates, sugars, lipids, nucleoproteins, glycoproteins,lipoproteins, steroids, natural products, etc.) that are commonly foundor produced in cells, whether the molecules themselves arenaturally-occurring or artificially created (e.g., by synthetic orrecombinant methods). For example, biomolecules include, but are notlimited to, enzymes, receptors, glycosaminoglycans, neurotransmitters,hormones, cytokines, cell response modifiers such as growth factors andchemotactic factors, antibodies, vaccines, haptens, toxins, interferons,ribozymes, anti-sense agents, plasmids, DNA, and RNA. Exemplary growthfactors include but are not limited to bone morphogenic proteins (BMP's)and their active fragments or subunits. In some embodiments, thebiomolecule is a growth factor, chemotactic factor, cytokine,extracellular matrix molecule, or a fragment or derivative thereof, forexample, a cell attachment sequence such as a peptide containing thesequence, RGD.

The term “tissue-derived material”, as used herein, refers to a materialthat is obtained from an animal tissue. A tissue-derived material mayinclude the tissue itself, a portion thereof, or one or more componentsthereof. For example, bone-derived tissue includes a whole bone, a boneparticle, and bone or bone pieces that have been processed to remove oneor more of cells, collagen, other extracellular matrix components,mineral, etc. In certain embodiments, tissue-derived material is treatedto removed any infectious agents, in particular, pathogens (e.g.,viruses, bacteria, fungi, parasites, etc.) In certain embodiments,tissue-derived material is treated to kill or remove any living cells orviruses. In certain particular embodiments, the tissue-derived materialincludes the extracellular matrix portion of a tissue. In certainembodiments, the tissue-derived material is purified extracellularmatrix.

The term “carbohydrate” refers to a sugar or polymer of sugars. Theterms “saccharide”, “polysaccharide”, “carbohydrate”, and“oligosaccharide”, may be used interchangeably. Most carbohydrates arealdehydes or ketones with many hydroxyl groups, usually one on eachcarbon atom of the molecule. Carbohydrates generally have the molecularformula C_(n)H_(2n)O_(n). A carbohydrate may be a monosaccharide, adisaccharide, trisaccharide, oligosaccharide, or polysaccharide. Themost basic carbohydrate is a monosaccharide, such as glucose, sucrose,galactose, mannose, ribose, arabinose, xylose, and fructose.Disaccharides are two joined monosaccharides. Exemplary disaccharidesinclude sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and six monosaccharide units(e.g., raffinose, stachyose), and polysaccharides include six or moremonosaccharide units. Exemplary polysaccharides include starch,glycogen, and cellulose. Carbohydrates may contain modified saccharideunits such as 2′-deoxyribose wherein a hydroxyl group is removed,2′-fluororibose wherein a hydroxyl group is replace with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose. (e.g.,2′-fluororibose, deoxyribose, and hexose). Carbohydrates may exist inmany different forms, for example, conformers, cyclic forms, acyclicforms, stereoisomers, tautomers, anomers, and isomers.

The term “composite” is used to refer to a unified combination of two ormore distinct materials. The composite may be homogeneous orheterogeneous. For example, a composite may be a combination ofbone-derived particles and a polymer; or a combination of a bonesubstitute material and a polymer. In certain embodiments, the compositehas a particular orientation.

“Demineralized” is used to refer to bone-derived material (e.g.,particles) that have been subjected to a process that causes a decreasein the original mineral content. As utilized herein, the phrase“superficially demineralized” as applied to bone particles refers tobone particles possessing at least about 90% by weight of their originalinorganic mineral content. The phrase “partially demineralized” asapplied to the bone particles refers to bone particles possessing fromabout 8% to about 90% by weight of their original inorganic mineralcontent, and the phrase “fully demineralized” as applied to the boneparticles refers to bone particles possessing less than about 8% byweight, for example, less than about 1% by weight, of their originalinorganic mineral content. The unmodified term “demineralized” asapplied to the bone particles is intended to cover any one orcombination of the foregoing types of demineralized bone particles.

“Deorganified”, as herein applied to matrices, particles, etc., refersto bone or cartilage matrices, particles, etc., that were subjected to aprocess that removes at least part of their original organic content. Insome embodiments, at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 99% of the organic content of the starting material is removed.Deorganified bone from which substantially all the organic componentshave been removed is termed “anorganic.”

The term “electromagnetic radiation” refers to a self-propagating wavewith both electric and magnetic components. The wave travels at thespeed of light through a vacuum. The magnetic and electric componentsoscillate at right angles to each other and also to the direction ofpropagation of the wave. Electromagnetic radiation is typicallyclassified according to the frequency of the wave. In order ofincreasing frequency, they are radio waves, microwaves, infrared (IR)radiation, visible light, ultraviolet (UV) radiation, X-rays, and gammarays. In certain contexts, electromagnetic radiation is referred to aslight.

As used herein, the term “flowable polymer material” refers to acomposition including one or more of monomers, pre-polymers, oligomers,low molecular weight polymers, uncross-linked polymers, partiallycross-linked polymers, partially polymerized polymers, polymers, orcombinations thereof that have been rendered formable. One skilled inthe art will recognize that the flowable polymer material need not be apolymer but may be polymerizable. In some embodiments, flowable polymermaterials include polymers that have been heated past their glasstransition or melting point. Alternatively or in addition, a flowablepolymer material may include partially polymerized polymer, telechelicpolymer, or prepolymer. A pre-polymer is a low molecular weight oligomertypically produced through step growth polymerization. The pre-polymeris formed with an excess of one of the components to produce moleculesthat are all terminated with the same group. For example, a diol and anexcess of a diisocyanate may be polymerized to produce isocyanateterminated prepolymer that may be combined with a diol to form apolyurethane. Alternatively or in addition, the flowable polymermaterial may be a polymer material/solvent mixture that sets when thesolvent is removed.

As used herein, “formable” materials are those that can be shaped bymechanical deformation. Exemplary methods of deformation include,without limitation, injection molding, extrusion, injection, pressing,casting, rolling, and molding.

As used herein, the term “glass transition temperature” (T_(g))indicates the lowest temperature at which an amorphous or partiallyamorphous polymer is considered softened and possibly flowable. Asreferred to herein, the value of T_(g) is to be determined usingdifferential calorimetry as per ASTM Standard E1356-98 “Standard TestMethod for Assignment of the Glass Transition Temperatures byDifferential Scanning calorimetry or Differential Thermal Analysis.”

As used herein, the term “melting temperature” (T_(m)) is defined as thetemperature, at atmospheric pressure, at which a polymer transitionsfrom a crystalline state to a viscous flow state. As referred to herein,the value of T_(m) is the value of T_(pm1) as determined according toper ASTM Standard D3418-99 “Standard Test Method for TransitionTemperatures of Polymers By Differential Scanning calorimetry.”

The term “mineralized” refers to bone-derived materials that have beensubjected to a process that caused a decrease in their original organiccontent (e.g., de-fatting, de-greasing). Such a process results in anincrease in the relative inorganic mineral content of the bone-derivedmaterial. Mineralization may also refer to the mineralization of amatrix such as extracellular matrix or demineralized bone matrix. Themineralization process may take either in vivo or in vitro.

“Non-demineralized”, as herein applied to bone or bone particles, refersto bone or bone-derived material (e.g., particles) that have not beensubjected to a demineralization process (i.e., a procedure that totallyor partially removes the original inorganic content of bone).

The term “osteoconductive”, as used herein, refers to the ability of asubstance or material to provide surfaces which are receptive to thegrowth of new bone.

The term “osteogenic” refers to the ability of a substance or materialthat can induce bone formation.

“Osteoinductive”, as used herein, refers to the quality of being able torecruit cells (e.g., osteoblasts) from the host that have the potentialto stimulate new bone formation. In general, osteoinductive materialsare capable of inducing heterotopic ossification, that is, boneformation in extraskeletal soft tissues (e.g., muscle).

The term “osteoimplant” is used herein in its broadest sense and is notintended to be limited to any particular shapes, sizes, configurations,compositions, or applications. Osteoimplant refers to any device ormaterial for implantation that aids or augments bone formation orhealing. Osteoimplants are often applied at a bone defect site, e.g.,one resulting from injury, defect brought about during the course ofsurgery, infection, malignancy, inflammation, or developmentalmalformation. Osteoimplants can be used in a variety of orthopedic,neurosurgical, dental, and oral and maxillofacial surgical proceduressuch as the repair of simple and compound fractures and non-unions,external, and internal fixations, joint reconstructions such asarthrodesis, general arthroplasty, deficit filling, disectomy,laminectomy, anterior cerival and thoracic operations, spinal fusions,etc.

The term “penetrate” refers to the ability of one substance to invade orinfiltrate another. Penetrate may refer to complete or partialpenetration. A polymer may infiltrate the particles of the composite.That is, the polymer may infiltrate the voids, gaps, holes, pores,crevices, etc. of the particles. After implantation, cells, tissue, orbone may invade the implanted composite.

The term “plasticizer”, as used herein, refers to an additive thatsoftens hard polymers or plastics. The plasticizer makes the polymerformable or flexible. Plasticizers are thought to work by embeddingthemselves between the chains of polymers, spacing them apart, and thuslowering the glass transition temperature. Preferably, the plasticizersused in the inventive composites are non-toxic and biocompatible. Incertain embodiments, as the plasticizer diffuses out of the compositeosteoimplant the composite loses its formability.

The terms “polynucleotide”, “nucleic acid”, or “oligonucleotide” referto a polymer of nucleotides. The terms “polynucleotide”, “nucleic acid”,and “oligonucleotide”, may be used interchangeably. Typically, apolynucleotide comprises at least three nucleotides. DNAs and RNAs areexemplary polynucleotides. The polymer may include natural nucleosides(i.e., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine),nucleoside analogs (e.g., 2-aminoadenosine, 2-thithymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine,C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemicallymodified bases, biologically modified bases (e.g., methylated bases),intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyriboses, arabinose, and hexose), or modified phosphate groups(e.g., phosphorothioates and 5′-N-phosphoramidite linkages). The polymermay also be a short strand of nucleic acids such as RNAi, siRNA, orshRNA.

As used herein, a “polypeptide”, “peptide”, or “protein” includes astring of at least three amino acids linked together by peptide bonds.The terms “polypeptide”, “peptide”, and “protein”, may be usedinterchangeably. In some embodiments, peptides may contain only naturalamino acids, although non-natural amino acids (i.e., compounds that donot occur in nature but that can be incorporated into a polypeptidechain) and/or amino acid analogs as are known in the art mayalternatively be employed. Also, one or more of the amino acids in apeptide may be modified, for example, by the addition of a chemicalentity such as a carbohydrate group, a phosphate group, a farnesylgroup, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. In oneembodiment, the modifications of the peptide lead to a more stablepeptide (e.g., greater half-life in vivo). These modifications mayinclude cyclization of the peptide, the incorporation of D-amino acids,etc. None of the modifications should substantially interfere with thedesired biological activity of the peptide.

The terms “polysaccharide” or “oligosaccharide”, as used herein, referto any polymer or oligomer of carbohydrate residues. The polymer oroligomer may consist of anywhere from two to hundreds to thousands ofsugar units or more. “Oligosaccharide” generally refers to a relativelylow molecular weight polymer, while “polysaccharide” typically refers toa higher molecular weight polymer. Polysaccharides may be purified fromnatural sources such as plants or may be synthesized de novo in thelaboratory. Polysaccharides isolated from natural sources may bemodified chemically to change their chemical or physical properties(e.g., reduced, oxidized, phosphorylated, cross-linked). Carbohydratepolymers or oligomers may include natural sugars (e.g., glucose,fructose, galactose, mannose, arabinose, ribose, xylose, etc.) and/ormodified sugars (e.g., 2′-fluororibose, 2′-deoxyribose, etc.).Polysaccharides may also be either straight or branched. They maycontain both natural and/or unnatural carbohydrate residues. The linkagebetween the residues may be the typical ether linkage found in nature ormay be a linkage only available to synthetic chemists. Examples ofpolysaccharides include cellulose, maltin, maltose, starch, modifiedstarch, dextran, poly(dextrose), and fructose. Glycosaminoglycans arealso considered polysaccharides. Sugar alcohol, as used herein, refersto any polyol such as sorbitol, mannitol, xylitol, galactitol,erythritol, inositol, ribitol, dulcitol, adonitol, arabitol,dithioerythritol, dithiothreitol, glycerol, isomalt, and hydrogenatedstarch hydrolysates.

The term “porogen” refers to a chemical compound that may be part of theinventive composite and upon implantation or prior to implantationdiffuses, dissolves, and/or degrades to leave a pore in the osteoimplantcomposite. The porogen may be introduced into the composite duringmanufacture, during preparation of the composite (e.g., in the operatingroom), or after implantation. The porogen essentially reserves space inthe composite while the composite is being molded but once the compositeis implanted the porogen diffuses, dissolves, or degrades, therebyinducing porosity into the composite. In this way the porogen provideslatent pores. In certain embodiments, the porogen may also be leachedout of the composite before implantation. This resulting porosity of theimplant generated during manufacture or after implantation (i.e.,“latent porosity”) is thought to allow infiltration by cells, boneformation, bone remodeling, osteoinduction, osteoconduction, and/orfaster degradation of the osteoimplant. A porogen may be a gas (e.g.,carbon dioxide, nitrogen, or other inert gas), liquid (e.g., water,biological fluid), or solid. Porogens are typically water soluble suchas salts, sugars (e.g., sugar alcohols), polysaccharides (e.g., dextran(poly(dextrose)), water soluble small molecules, etc. Porogen can alsobe natural or synthetic polymers, oligomers, or monomers that are watersoluble or degrade quickly under physiological conditions. Exemplarypolymers include polyethylene glycol, poly(vinylpyrollidone), pullulan,poly(glycolide), poly(lactide), poly(lactide-co-glycolide), otherpolyesters, and starches.

The term “porosity” refers to the average amount of non-solid spacecontained in a material (e.g., a composite of the present invention).The porosity of a composite can be defined as the ratio of the totalvolume of the pores (i.e., void volume) in the material to the overallvolume of the composite. Porosity may in certain embodiments refer to“latent porosity” wherein pores are only formed upon diffusion,dissolution, or degradation of a material occupying the pores. The poresin such an instance may be formed after implantation.

As used herein, the term “remodeling” describes the process by whichnative bone, processed bone allograft, whole bone sections employed asgrafts, and other bony tissues are replaced with new cell-containinghost bone tissue by the action of osteoclasts and osteoblasts.Remodeling also describes the process by which non-bony native tissueand tissue grafts are removed and replaced with new, cell-containingtissue in vivo. Remodeling also describes how inorganic materials (e.g.,calcium-phosphate materials, such as β-tricalcium phosphate) is replacedwith living bone.

As used herein, the term “settable” refers to a material that may berendered more resistant to mechanical deformation as compared to aformable state.

As used herein, the term “set” refers to the state of a material thathas been rendered more resistant to mechanical deformation with respectto a formable state.

The term “shaped” as used to characterize a material (e.g., composite)or an osteoimplant refers to a material or osteoimplant of a determinedor regular form or configuration in contrast to an indeterminate orvague form or configuration (as in the case of a lump or other solidmatrix of special form). The material may be shaped into any shape,configuration, or size. Materials can be shaped as sheets, blocks,plates, disks, cones, pins, screws, tubes, teeth, bones, portions ofbones, wedges, cylinders, threaded cylinders, and the like, as well asmore complex geometric configurations.

As used herein, the term “small molecule” is used to refer to molecules,whether naturally-occurring or artificially created (e.g., via chemicalsynthesis), that have a relatively low molecular weight. Typically,small molecules have a molecular weight of less than about 2,500 g/mol,more preferably less than 1000 g/mol. Preferred small molecules arebiologically active in that they produce a local or systemic effect inanimals, preferably mammals, more preferably humans. In certainpreferred embodiments, the small molecule is a drug. Preferably, thoughnot necessarily, the drug is one that has already been deemed safe andeffective for use by an appropriate governmental agency or body (e.g.,the U.S. Food and Drug Administration).

As used herein, the term “transformation” describes the process by whicha material is removed from an implant site and replaced by host tissueafter implantation. Transformation may be accomplished by a combinationof processes, including but not limited to remodeling, degradation,resorption, and tissue growth and/or formation. Removal of the materialmay be cell-mediated or accomplished through chemical processes, such asdissolution and hydrolysis.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The present invention stems from the recognition that it would be usefulto have bone substitute material that is sufficiently moldable orflowable to administer to a subject by injecting or molding thecomposite into an irregularly shaped implantation site (e.g., a bonedefect, a void, or a fracture). The composite may be made moldable orflowable before administration such as by heating the composite orcombining the composite with a suitable solvent. The viscosity of theresulting composite may range from a thick, flowable liquid (forexample, like molasses or honey) to a moldable, dough-like putty. Incertain embodiments, the composite is naturally moldable or flowable andis set by exposing the composite to predetermined conditions (e.g.,cooling, UV irradiation, IR irradiation, microwave irradiation). Theinvention also provides methods of preparing and using the inventivecomposite as well as kits for administering the inventive composite. Inone embodiment, bone-derived tissue or other particulate material iscombined with a polymer and injected, extruded, molded, or similarlydelivered to a tissue site (e.g., bony defect) of a subject. Theinventive composite is engineered to set in situ to form a solidcomposite that has a desired mechanical strength. In certainembodiments, the polymer may include monomers or pre-polymers, or it maybe a polymer that has been rendered formable by heating it above itsglass transition temperature or melting point, or through combinationwith a solvent.

Particulate Component of Composite

Bone-Derived Material

Any type of particles comprising inorganic material, bone substitutematerial, bone-derived material, or combinations or composites thereofmay be utilized in the present invention. In certain embodiments, abone-derived material is used in the inventive composites. In oneembodiment, bone particles employed in the preparation of the boneparticle-containing composite are obtained from cortical, cancellous,and/or corticocancellous bone. The bone-derived material may be derivedfrom any vertebrate. The bone-derived material may be of autogenous,allogenic, and/or xenogeneic origin. In certain embodiments, thebone-derived material is autogenous, that is, the bone-derived materialis from the subject being treated. In other embodiments, thebone-derived material is allogenic (e.g., from donors). Preferably, thesource of the bone is matched to the eventual recipient of the inventivecomposite (i.e., the donor and recipient are preferably of the samespecies). For example, human bone-derived material is typically used ina human subject. In certain particular embodiments, the bone particlesare obtained from cortical bone of allogenic origin. In certainembodiments, the bone-derived material is obtained from bone ofxenogeneic origin. Porcine and bovine bone are particularly advantageoustypes of xenogeneic bone tissue that can be used individually or incombination as sources for the bone-derived material. Xenogenic bonetissue may be combined with allogenic or autogenous bone.

Particles of bone-derived material are formed by any process known tobreak down bone into small pieces. Exemplary processes for forming suchparticles include milling whole bone to produce fibers, chipping wholebone, cutting whole bone, grinding whole bone, fracturing whole bone inliquid nitrogen, or otherwise disintegrating the bone tissue. Particlescan optionally be sieved to produce particles of a specific size range.The particles may be of any shape or size. Exemplary shapes includespheroidal, plates, fibers, cuboidal, sheets, rods, oval, strings,elongated particles, wedges, discs, rectangular, polyhedral, etc. Insome embodiments, particles may be between about 10 microns and about1000 microns in diameter or more. In some embodiments, particles may bebetween about 20 microns and about 800 microns in diameter or more. Incertain embodiments, the particles range in size from approximately 100microns in diameter to approximately 500 microns in diameter. In certainembodiments, the particles range in size from approximately 300 micronsin diameter to approximately 800 microns in diameter. As for irregularlyshaped particles, the recited dimension ranges may represent the lengthof the greatest or smallest dimension of the particle. As will beappreciated by one of skill in the art, for injectable composites, themaximum particle size will depend in part on the size of the cannula orneedle through which the material will be delivered

In certain embodiments, the particle size distribution of the particlesthat are combined with a polymer to form the inventive composite withrespect to a mean value may be plus or minus, e.g., about 10% or less ofthe mean value, about 20% or less of the mean value, about 30% or lessof the mean value, about 40% or less of the mean value, about 50% orless of the mean value, about 60% or less of the mean value, about 70%or less of the mean value, about 80% or less of the mean value, or about90% or less of the mean value. In other embodiments, the particle sizedistribution of the particles that are combined with a polymer to formthe inventive composite with respect to a median value may be plus orminus, e.g., about 10% or less of the median value, about 20% or less ofthe median value, about 30% or less of the median value, about 40% orless of the median value, about 50% or less of the median value, about60% or less of the median value, about 70% or less of the median value,about 80% or less of the median value, or about 90% or less of themedian value. In certain embodiments, at least about 60, 70, or 80weight percent of the particles posses a median length of about 10microns to about 1000 microns in their greatest dimension. In certainembodiments, at least about 60, 70, or 80 weight percent of theparticles posses a median length of about 20 microns to about 800microns in their greatest dimension. For particles that are fibers orother elongated particles, at least about 60 weight percent, at leastabout 70 weight percent, or at least about 80 weight percent of theparticles possess a median length of from about 2 to about 200 mm, ormore preferably from about 10 to about 100 mm, a median thickness offrom about 0.05 to about 2 mm, and preferably from about 0.2 to about 1mm, and a median width of from about 1 mm to about 20 mm and preferablyfrom about 2 to about 5 mm. The particles may possess a median length tomedian thickness ratio from at least about 5:1 up to about 500:1,preferably from at least about 50:1 up to about 500:1, or more andpreferably from about 50:1 up to about 100:1; and a median length tomedian width ratio of from about 10:1 to about 200:1 and preferably fromabout 50:1 to about 100:1. In certain embodiments, the bone-derivedparticles are short fibers having a cross-section of about 300 micronsto about 100 microns and a length of about 1 mm to about 4 mm.

The processing of the bone to provide the particles may be adjusted tooptimize for the desired size and/or distribution of the particles. Thedesired properties of the resulting inventive composite (e.g.,mechanical properties) may also be engineered by adjusting the weightpercent, shapes, sizes, distribution, etc. of the bone-derived particlesor other particles. For example, an inventive composite may be made moreviscous by including a higher percentage of particles.

The bone-derived particles utilized in accordance with the presentinvention may be demineralized, non-demineralized, mineralized, oranorganic. In certain embodiments, the resulting bone-derived particlesare used “as is” in preparing the inventive composites. In otherembodiments, the particles are defatted and disinfected. An exemplarydefatting/disinfectant solution is an aqueous solution of ethanol. Otherorganic solvent may also be used in the defatting and disinfecting theparticles. For example, methanol, isopropanol, butanol, DMF, DMSO,diethyl ether, hexanes, glyme, tetrahydrofuran, chloroform, methylenechloride, and carbon tetrachloride may be used. In certain embodiments,a non-halogenated solvent is used. The defatting/disinfecant solutionmay also include a detergent (e.g., an aqueous solution of a detergent).Ordinarily, at least about 10 to about 40 percent by weight of water(i.e., about 60 to about 90 weight percent of defatting agent such asalcohol) should be present in the defatting/disinfecting solution toproduce optimal lipid removal and disinfection within the shortestperiod of time. An exemplary concentration range of the defattingsolution is from about 60 to about 85 weight percent alcohol, forexample, about 70 weight percent alcohol.

In certain embodiments, the particles are demineralized. Thebone-derived particles are optionally demineralized in accordance withknown and/or conventional procedures in order to reduce their inorganicmineral content. Demineralization methods remove the inorganic mineralcomponent of bone by employing acid solutions. Such methods are wellknown in the art, see for example, Reddi, et al., Proc. Nat. Acad. Sci.,1972, 69:1601-1605, the contents of which are incorporated herein byreference. The strength of the acid solution, the shape and dimensionsof the bone particles and the duration of the demineralization treatmentwill determine the extent of demineralization. Reference in this regardis made to Lewandrowski, et al., J. Biomed. Mater. Res., 1996,31:365-372 and U.S. Pat. No. 5,290,558, the contents of both of whichare incorporated herein by reference.

In an exemplary defatting/disinfecting/demineralization procedure, thebone particles are subjected to a defatting/disinfecting step, followedby an acid demineralization step. An exemplary defatting/disinfectantsolution is an aqueous solution of ethanol. Ordinarily, at least about10 to about 40 percent by weight of water (i.e., about 60 to about 90weight percent of defatting agent such as alcohol) should be present inthe defatting/disinfecting solution to produce optimal lipid removal anddisinfection within a reasonable period of time. An exemplaryconcentration range of the defatting solution is from about 60 to about85 weight percent alcohol, for example, about 70 weight percent alcohol.Ethanol is typically the alcohol used in this step; however, otheralcohols such as methanol, propanol, isopropanol, denatured ethanol,etc. may also be used. Following defatting, the bone particles areimmersed in acid over time to effect their demineralization. The acidalso disinfects the bone by killing viruses, vegetative microorganisms,and spores. Acids which can be employed in this step include inorganicacids such as hydrochloric acid and organic acids such as peraceticacid. After acid treatment, the demineralized bone particles are rinsedwith sterile water to remove residual amounts of acid and thereby raisethe pH. The bone particles may be dried, for example, by lyophilization,before being incorporated into the composite. The bone particles may bestored under aseptic conditions, for example, in a lyophilized state,until they are used or sterilized using known methods (e.g., gammairradiation) shortly before combining them with a polymer.

As utilized herein, the phrase “superficially demineralized” as appliedto the bone particles refers to bone particles possessing at least about90% by weight of their original inorganic mineral content. The phrase“partially demineralized” as applied to the bone particles refers tobone particles possessing from about 8% to about 90% weight of theiroriginal inorganic mineral content, and the phrase “fully demineralized”as applied to the bone particles refers to bone particles possessingless than about 8%, preferably less than about 1%, by weight of theiroriginal inorganic mineral content. The unmodified term “demineralized”as applied to the bone particles is intended to cover any one orcombination of the foregoing types of demineralized bone particles, thatis, superficially demineralized, partially demineralized, or fullydemineralized bone particles.

In an alternative embodiment, surfaces of bone particles may be lightlydemineralized according to the procedures in our commonly owned U.S.patent application Ser. No. 10/285,715, filed Nov. 1, 2002, published asU.S. Patent Publication No. 2003/0144743, on Jul. 31, 2003, the contentsof which are incorporated herein by reference. Even minimaldemineralization, for example, of less than 5% removal of the inorganicphase, increases the hydroxylation of bone fibers and the surfaceconcentration of amine groups. Demineralization may be so minimal, forexample, less than 1%, that the removal of the calcium phosphate phaseis almost undetectable. Rather, the enhanced surface concentration ofreactive groups defines the extent of demineralization. This may bemeasured, for example, by titrating the reactive groups. In oneembodiment, in a polymerization reaction that utilizes the exposedallograft surfaces to initiate a reaction, the amount of unreactedmonomer remaining may be used to estimate reactivity of the surfaces.Surface reactivity may be assessed by a surrogate mechanical test, suchas a peel test of a treated coupon of bone adhering to a polymer.

In certain embodiments, the bone-derived particles are subjected to aprocess that partially or totally removes their initial organic contentto yield mineralized and anorganic bone particles, respectively.Different mineralization methods have been developed and are known inthe are (Hurley et al., Milit. Med. 1957, 101-104; Kershaw, Pharm. J.6:537, 1963; and U.S. Pat. No. 4,882,149; each of which is incorporatedherein by reference). For example, a mineralization procedure caninclude a de-greasing step followed by a basic treatment (with ammoniaor another amine) to degrade residual proteins and a water washing (U.S.Pat. Nos. 5,417,975 and 5,573,771; both of which are incorporated hereinby reference). Another example of a mineralization procedure includes adefatting step where bone particles are sonicated in 70% ethanol for 1-3hours.

If desired, the bone-derived particles can be modified in one or moreways, e.g., their protein content can be augmented or modified asdescribed, for example, in U.S. Pat. Nos. 4,743,259 and 4,902,296, thecontents of both of which are incorporated herein by reference.

Mixtures or combinations of one or more of the foregoing types ofbone-derived particles can be employed. For example, one or more of theforegoing types of demineralized bone-derived particles can be employedin combination with non-demineralized bone-derived particles, i.e.,bone-derived particles that have not been subjected to ademineralization process, or inorganic materials. The amount of eachindividual type of bone-derived particle employed can vary widelydepending on the mechanical and biological properties desired. Thus,mixtures of bone-derived particles of various shapes, sizes, and/ordegrees of demineralization may be assembled based on the desiredmechanical, thermal, chemical, and biological properties of thecomposite. A desired balance between the various properties of thecomposite (e.g., a balance between mechanical and biological properties)may be achieved by using different combinations of particles. Suitableamounts of various particle types can be readily determined by thoseskilled in the art on a case-by-case basis by routine experimentation.

The differential in strength, osteogenicity, and other propertiesbetween partially and fully demineralized bone-derived particles on theone hand, and non-demineralized, superficially demineralizedbone-derived particles, inorganic ceramics, and bone substitutes on theother hand can be exploited. For example, in order to increase thecompressive strength of the osteoimplant, the ratio of nondemineralizedand/or superficially demineralized bone-derived particles to partiallyor fully demineralized bone-derived particles may favor the former, andvice versa. The bone-derived particles in the composite also play abiological role. Non-demineralized bone-derived particles bring aboutnew bone in-growth by osteoconduction. Demineralized bone-derivedparticles likewise play a biological role in bringing about new bonein-growth by osteoinduction. Both types of bone-derived particles aregradually remodeled and replaced by new host bone as degradation of thecomposite progresses over time. Thus, the use of various types of boneparticles can be used to control the overall mechanical and biologicalproperties, i.e., the strength, osteoconductivity, and/orosteoinductivity, etc., of the osteoimplant.

Surface Modification of Bone-Derived Particles

The bone-derived particles may be optionally treated to enhance theirinteraction with the polymer of the composite or to confer some propertyto the particle surface. While some bone-derived particles will interactreadily with the monomer and be covalently linked to the polymer matrix,it may be desirable to modify the surface of the bone-derived particlesto facilitate incorporation into polymers that do not bond well to bone,such as poly(lactides). Surface modification may provide a chemicalsubstance that is strongly bonded to the surface of the bone, e.g.,covalently bonded to the surface. The bone-derived particles may also becoated with a material to facilitate interaction with the polymer of thecomposite.

In one embodiment, silane coupling agents are employed to link a monomeror initiator molecule to the surface of the bone-derived particles. Thesilane has at least two sections, a set of three leaving groups and anactive group. The active group may be connected to the silicon atom inthe silane by an elongated tether group. An exemplary silane couplingagent is 3-trimethoxysilylpropylmethacrylate, available from UnionCarbide. The three methoxy groups are the leaving groups, and themethacrylate active group is connected to the silicon atom by a propyltether group. In one embodiment, the leaving group is an alkoxy groupsuch as methoxy or ethoxy. Depending on the solvent used to link thecoupling agent to the bone-derived particle, hydrogen or alkyl groupssuch as methyl or ethyl may serve as the leaving group. The length ofthe tether determines the intimacy of the connection between the polymermatrix and the bone-derived particle. By providing a spacer between thebone-derived particle and the active group, the tether also reducescompetition between chemical groups at the particle surface and theactive group and makes the active group more accessible to the monomerduring polymerization.

In one embodiment, the active group is an analog of the monomer of thepolymer used in the composite. For example, amine active groups will beincorporated into polyamides, polyesters, polyurethanes, polycarbonates,polycaprolactone, and other polymer classes based on monomers that reactwith amines, even if the polymer does not contain an amine.Hydroxy-terminated silanes will be incorporated into polyamino acids,polyesters, polycaprolactone, polycarbonates, polyurethanes, and otherpolymer classes that include hydroxylated monomers. Aromatic activegroups or active groups with double bonds will be incorporated intovinyl polymers and other polymers that grow by radical polymerization(e.g., polyacrylates, polymethacrylates). It is not necessary that theactive group be monofunctional. Indeed, it may be preferable that activegroups that are to be incorporated into polymers via step polymerizationbe difunctional. A silane having two amines, even if one is a secondaryamine, will not terminate a polymer chain but can react with ends of twodifferent polymer chains. Alternatively, the active group may bebranched to provide two reactive groups in the primary position.

An exemplary list of silanes that may be used with the invention isprovided in U.S. Patent Publication No. 2004/0146543, the contents ofwhich are incorporated herein by reference. Silanes are available fromcompanies such as Union Carbide, AP Resources Co. (Seoul, South Korea),and BASF. Where the silane contains a potentially non-biocompatiblemoiety as the active group, it should be used to tether a biocompatiblecompound to the bone particle using a reaction in which thenon-biocompatible moiety is the leaving group. It may be desirable toattach the biocompatible compound to the silane before attaching thesilane to the bone-derived particle, regardless of whether the silane isbiocompatible or not. The derivatized silanes may be mixed with silanesthat can be incorporated directly into the polymer and reacted with thebone-derived particles, coating the bone particles with a mixture of“bioactive” silanes and “monomer” silanes. U.S. Pat. No. 6,399,693, thecontents of which are incorporated herein by reference disclosescomposites of silane modified polyaromatic polymers and bone.Silane-derivatized polymers may be used in the inventive compositesinstead of or in addition to first silanizing the bone-derivedparticles.

The active group of the silane may be incorporated directly into thepolymer or may be used to attach a second chemical group to the boneparticle. For example, if a particular monomer polymerizes through afunctional group that is not commercially available as a silane, themonomer may be attached to the active group.

Non-silane linkers may also be employed to produce composites accordingto the invention. For example, isocyanates will form covalent bonds withhydroxyl groups on the surface of hydroxyapatite ceramics (de Wijn, etal., “Grafting PMMA on Hydroxyapatite Powder Particles usingIsocyanatoethylmethacrylate,” Fifth World Biomaterials Congress, May29-Jun. 2, 1996, Toronto, CA). Isocyanate anchors, with tethers andactive groups similar to those described with respect to silanes, may beused to attach monomer-analogs to the bone particles or to attachchemical groups that will link covalently or non-covalently with apolymer side group. Polyamines, organic compounds containing one or moreprimary, secondary, or tertiary amines, will also bind with both thebone particle surface and many monomer and polymer side groups.Polyamines and isocyanates may be obtained from Aldrich.

Alternatively, a biologically active compound such as a biomolecule, asmall molecule, or a bioactive agent may be attached to the bone-derivedparticle through the linker. For example, mercaptosilanes will reactwith the sulfur atoms in proteins to attach them to the bone-derivedparticle. Aminated, hydroxylated, and carboxylated silanes will reactwith a wide variety functional groups. Of course, the linker may beoptimized for the compound being attached to the bone-derived particle.

Biologically active molecules can modify non-mechanical properties ofthe composite as it is degraded. For example, immobilization of a drugon the bone particle allows it to be gradually released at an implantsite as the composite is degraded. Anti-inflammatory agents embeddedwithin the composite will control the inflammatory response long afterthe initial response to injection of the composite. For example, if apiece of the composite fractures several weeks after injection,immobilized compounds will reduce the intensity of any inflammatoryresponse, and the composite will continue to degrade through hydrolyticor physiological processes. Compounds may also be immobilized on thebone-derived particles that are designed to elicit a particularmetabolic response or to attract cells to the injection site.

Some biomolecules, small molecules, and bioactive agents may also beincorporated into the polymer used in the composite. For example, manyamino acids have reactive side chains. The phenol group on tyrosine hasbeen exploited to form polycarbonates, polyarylates, andpolyiminocarbonates (see Pulapura, et al., “Tyrosine-derivedpolycarbonates: Backbone-modified “pseudo”-poly(amino acids) designedfor biomedical applications,” Biopolymers, 1992, 32: 411-417; andHooper, et al., “Diphenolic monomers derived from the natural amino acidα-L-tyrosine: an evaluation of peptide coupling techniques,” J.Bioactive and Compatible Polymers, 1995, 10:327-340, the entire contentsof both of which are incorporated herein by reference). Amino acids suchas lysine, arginine, hydroxylysine, proline, and hydroxyproline alsohave reactive groups and are essentially tri-functional. Amino acidssuch as valine, which has an isopropyl side chain, are stilldifunctional. Such amino acids may be attached to the silane and stillleave one or two active groups available for incorporation into apolymer.

Non-biologically active materials may also be attached to the boneparticles. For example, radioopaque, luminescent, or magnetically activeparticles may be attached to the bone particles using the techniquesdescribed above. If a material, for example, a metal atom or cluster,cannot be produced as a silane or other group that reacts with calciumphosphate ceramics, then a chelating agent may be immobilized on thebone particle surface and allowed to form a chelate with the atom orcluster. As the bone is resorbed, these non-biodegradable materials arestill removed from the tissue site by natural metabolic processes,allowing the degradation of the polymer and the resorption of thebone-derived particles to be tracked using standard medical diagnostictechniques.

In an alternative embodiment, the bone-derived particle surface ischemically treated before being derivatized or combined with a polymer.For example, non-demineralized bone-derived particles may be rinsed withphosphoric acid, e.g., for 1 to 15 minutes in a 5-50% solution byvolume. Those skilled in the art will recognize that the relative volumeof bone particles and phosphoric acid solution (or any other solutionused to treat the bone particles), may be optimized depending on thedesired level of surface treatment. Agitation will also increase theuniformity of the treatment both along individual particles and acrossan entire sample of particles. The phosphoric acid solution reacts withthe mineral component of the bone to coat the particles with calciumphosphate, which may increase the affinity of the surface for inorganiccoupling agents such as silanes and for the polymer component of thecomposite. As noted above, the surface may be partially demineralized toexpose the collagen fibers at the particle surface.

The collagen fibers exposed by demineralization are typically relativelyinert but have some exposed amino acid residues that can participate inreactions. The collagen may be rendered more reactive by fraying thetriple helical structure of the collagen to increase the exposed surfacearea and the number of exposed amino acid residues. This not onlyincreases the surface area available for chemical reactions but also formechanical interaction with the polymer as well. Rinsing the partiallydemineralized bone particles in an alkaline solution will fray thecollagen fibrils. For example, bone particles may be suspended in waterat a pH of about 10 for about 8 hours, after which the solution isneutralized. One skilled in the art will recognize that this time periodmay be increased or decreased to adjust the extent of fraying.Agitation, for example, in an ultrasonic bath, may reduce the processingtime. Alternatively, the particles may be sonicated with water,surfactant, alcohol, or some combination of these.

Alternatively, the collagen fibers may be cross-linked. A variety ofcross-linking techniques suitable for medical applications are wellknown in the art (see, for example, U.S. Pat. No. 6,123,781, thecontents of which are incorporated herein by reference). For example,compounds like 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride, either alone or in combination with N-hydroxysuccinimide(NHS) will crosslink collagen at physiologic or slightly acidic pH(e.g., in pH 5.4 MES buffer). Acyl azides and genipin, a naturallyoccurring bicyclic compound including both carboxylate and hydroxylgroups, may also be used to cross-link collagen chains (see Simmons, etal, “Evaluation of collagen cross-linking techniques for thestabilization of tissue matrices,” Biotechnol. Appl. Biochem., 1993,17:23-29; PCT Publication WO98/19718, the contents of both of which areincorporated herein by reference). Alternatively, hydroxymethylphosphine groups on collagen may be reacted with the primary andsecondary amines on neighboring chains (see U.S. Pat. No. 5,948,386, theentire contents of which are incorporated herein by reference). Standardcross-linking agents such as mono- and dialdehydes, polyepoxy compounds,tanning agents including polyvalent metallic oxides, organic tannins,and other plant derived phenolic oxides, chemicals for esterification orcarboxyl groups followed by reaction with hydrazide to form activatedacyl azide groups, dicyclohexyl carbodiimide and its derivatives andother heterobifunctional crosslinking agents, hexamethylenediisocyanate, and sugars may also be used to cross-link the collagen.The bone-derived particles are then washed to remove all leachabletraces of the material. Enzymatic cross-linking agents may also be used.Additional cross-linking methods include chemical reaction, irradiation,application of heat, dehydrothermal treatment, enzymatic treatment, etc.One skilled in the art will easily be able to determine the optimalconcentrations of cross-linking agents and incubation times for thedesired degree of cross-linking.

Both frayed and unfrayed collagen fibers may be derivatized withmonomer, pre-polymer, oligomer, polymer, initiator, and/or biologicallyactive or inactive compounds, including but not limited to biomolecules,bioactive agents, small molecules, inorganic materials, minerals,through reactive amino acids on the collagen fiber such as lysine,arginine, hydroxylysine, proline, and hydroxyproline. Monomers that linkvia step polymerization may react with these amino acids via the samereactions through which they polymerize. Vinyl monomers and othermonomers that polymerize by chain polymerization may react with theseamino acids via their reactive pendant groups, leaving the vinyl groupfree to polymerize. Alternatively, or in addition, bone-derivedparticles may be treated to induce calcium phosphate deposition andcrystal formation on exposed collagen fibers. Calcium ions may bechelated by chemical moieties of the collagen fibers, and/or calciumions may bind to the surface of the collagen fibers. James et al.,Biomaterials 20:2203-2313, 1999; incorporated herein by reference. Thecalcium ions bound to the to the collagen provides a biocompatiblesurface, which allows for the attachment of cells as well as crystalgrowth. The polymer will interact with these fibers, increasinginterfacial area and improving the wet strength of the composite.

Additionally or alternatively, the surface treatments described above ortreatments such as etching may be used to increase the surface area orsurface roughness of the bone-derived particles. Such treatmentsincrease the interfacial strength of the particle/polymer interface byincreasing the surface area of the interface and/or the mechanicalinterlocking of the bone-derived particles and the polymer. Such surfacetreatments may also be employed to round the shape or smooth the edgesof bone particles to facilitate delivery of the inventive composite.Such treatment is particularly useful for injectable composites.

In some embodiments, surface treatments of the bone-derived particlesare optimized to enhance covalent attractions between the bone-derivedparticles and the polymer of the composite. In an alternativeembodiment, the surface treatment may be designed to enhancenon-covalent interactions between the bone-derived particle and thepolymer matrix. Exemplary non-covalent interactions includeelectrostatic interactions, hydrogen bonding, pi-bond interactions,hydrophobic interactions, van der Waals interactions, and mechanicalinterlocking. For example, if a protein or a polysaccharide isimmobilized on the bone-derived particle, the chains of the polymer willbecome physically entangled with the long chains of the biologicalpolymer when they are combined. Charged phosphate sites on the surfaceof the particles, produced by washing the bone particles in basicsolution, will interact with the amino groups present in manybiocompatible polymers, especially those based on amino acids. Thepi-orbitals on aromatic groups immobilized on a bone-derived particlewill interact with double bonds and aromatic groups of the polymer.

Additional Particulate Materials

Inorganic materials, including, but not limited, calcium phosphatematerials and bone substitute materials, may also be exploited for useas particulate inclusions in the inventive composites. Exemplaryinorganic inorganics for use with the invention include aragonite,dahlite, calcite, amorphous calcium carbonate, vaterite, weddellite,whewellite, struvite, urate, ferrihydrite, francolite, monohydrocalcite,magnetite, goethite, dentin, calcium carbonate, calcium sulfate, calciumphosphosilicate, sodium phosphate, calcium aluminate, calcium phosphate,hydroxyapatite, α-tricalcium phosphate, dicalcium phosphate,β-tricalcium phosphate, tetracalcium phosphate, amorphous calciumphosphate, octacalcium phosphate, and BIOGLASS™, a calcium phosphatesilica glass available from U.S. Biomaterials Corporation. Substitutedcalcium phosphate phases are also contemplated for use with theinvention, including but not limited to fluorapatite, chlorapatite,magnesium-substituted tricalcium phosphate, and carbonatehydroxyapatite. In certain embodiments, the inorganic material is asubstituted form of hydroxyapatite. For example, the hydroxyapatite maybe substituted with other ions such as fluoride, chloride, magnesium,sodium, potassium, etc. Additional calcium phosphate phases suitable foruse with the invention include those disclosed in U.S. Pat. RE 33,161and RE 33,221 to Brown et al.; U.S. Pat. Nos. 4,880,610; 5,034,059;5,047,031; 5,053,212; 5,129,905; 5,336,264; and 6,002,065 to Constantzet al.; U.S. Pat. Nos. 5,149,368; 5,262,166 and 5,462,722 to Liu et al.;U.S. Pat. Nos. 5,525,148 and 5,542,973 to Chow et al., U.S. Pat. Nos.5,717,006 and 6,001,394 to Daculsi et al., U.S. Pat. No. 5,605,713 toBoltong et al., U.S. Pat. No. 5,650,176 to Lee et al., and U.S. Pat. No.6,206,957 to Driessens et al, and biologically-derived or biomimeticmaterials such as those identified in Lowenstam H A, Weiner S, OnBiomineralization, Oxford University Press, 1989; each of which isincorporated herein by reference.

In another embodiment, a particulate composite material may be employedin the mixture with the polymer. For example, inorganic materials suchas those described above or bone-derived materials may be combined withproteins such as bovine serum albumin (BSA), collagen, or otherextracellular matrix components to form a composite. Alternatively or inaddition, inorganic materials or bone-derived materials may be combinedwith synthetic or natural polymers to form a composite using thetechniques described in our co-pending U.S. patent applications, U.S.Ser. No. 10/735,135, filed Dec. 12, 2003; U.S. Ser. No. 10/681,651,filed Oct. 8, 2003; and U.S. Ser. No. 10/639,912, filed Aug. 12, 2003,the contents of all of which are incorporated herein by reference. Thesecomposites may be partially demineralized as described herein to exposethe organic material at the surface of the composite before they arecombined with a polymer.

In certain embodiments, the particular composite material is onedescribed in U.S. patent applications, U.S. Ser. No. 10/771,736, filedFeb. 2, 2004, and published as US 2005/0027033; and U.S. Ser. No.11/336,127, filed Jan. 19, 2006, and published as US 2006/0216323; eachof which is incorporated herein by reference. Composite materialsdescribed in these applications include a polyurethane matrix and areinforcement embedded in the matrix. The polyurethane matrix may beformed by reaction of a polyisocyanate (e.g., lysine diisocyanate,toluene diisocyanate, arginine diisocyanate, asparagine diisocyanate,glutamine diisocyanate, hexamethylene diisocyanate, hexane diisocyanate,methylene bis-p-phenyl diisocyanate, isocyanurate polyisocyanates,1,4-butane diisocyanate, uretdione polyisocyanate, or aliphatic,alicyclic, or aromatic polyisocyanates) with an optionally hydroxylatedbiomolecule (e.g., a phospholipids, fatty acid, cholesterol,polysaccharide, starch, or a combination or modified form of any of theabove) to form a biodegradable polymer, while the reinforcementcomprises bone-derived material or a bone substitute (e.g., calciumcarbonate, calcium sulfate, calcium phosphosilicate, sodium phosphate,calcium aluminate, calcium phosphate, calcium carbonate, hydroxyapatite,demineralized bone, mineralized bone, or combinations or modified formsof any of these).

Particles of composite material for use in the present invention maycontain between about 5 and about 80% of bone-derived or other inorganicmaterial, for example, between about 60% and about 75%. Particulatematerials for use in the inventive composites may be modified toincrease the concentration of nucleophilic groups (e.g., amino orhydroxyl groups) at their surfaces using the techniques describedherein.

The inventive composite may contain between about 5% and 80% by weightbone-derived particles, bone substitute particles, or inorganic materialparticles. In certain embodiments, the particles make up between about10% and about 30% by weight of the composite. In certain embodiments,the particles make up between about 30% and about 50% by weight of thecomposite. In certain embodiments, the particles make up between about40% and about 50% by weight of the composite. In certain embodiments,the particles make up between about 60% and about 75% by weight of thecomposite. In certain embodiments, the particles make up between about45% and about 70% by weight of the composite. In certain embodiments,the particles make up between about 50% and about 65% by weight of thecomposite. In certain particular embodiments, the particles make upapproximately 20%, 25%, 30%, or 40% by weight of the composite. Incertain particular embodiments, the particles make up approximately 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, or 65% by weight of the composite.

Combining the Particles with a Polymer

To form the inventive composite, the particles as discussed herein arecombined with a polymer thereby forming a naturally moldable or flowablecomposite or a composite that can be made moldable or flowable. Thepolymer may be further modified by further cross-linking orpolymerization after combination with particles to form a composite inwhich the polymer is covalently linked to the particles. In someembodiments, the polymer is a polymer/solvent mixture that hardens whenthe solvent is removed (e.g., when the solvent is allowed to evaporateor diffuse away). Exemplary solvents include but are not limited toalcohols (e.g., methanol, ethanol, propanol, butanol, hexanol, etc.),water, saline, DMF, DMSO, glycerol, and PEG. In certain embodiments, thesolvent is a biological fluid such as blood, plasma, serum, marrow, etc.In certain embodiments, the inventive composite is heated above themelting or glass transition temperature of one or more of its componentsand becomes set after implantation as it cools. In certain embodiments,the inventive composite is set by exposing the composite to a heatsource, or irradiating it with microwaves, IR rays, or UV light. Theparticles may also be mixed with a polymer that is sufficiently pliableto combine with the particles but that may require further treatment,for example, combination with a solvent or heating, to become a flowableor moldable composite.

In some embodiments, the composite is produced with a flowable polymerand then set in situ. For example, the cross-link density of a lowmolecular weight polymer may be increased by exposing it toelectromagnetic radiation (e.g., UV light) or an alternative energysource. Alternatively, a photoactive cross-linking agent, chemicalcross-linking agent, additional monomer, or combinations thereof may bemixed into the composite. Exposure to UV light after the mixture isinjected into the implant site will increase one or both of themolecular weight and cross-link density, stiffening the polymer andthereby the composite. The polymer component of the composite may alsobe softened by a solvent, e.g., ethanol. If a biocompatible solvent isused, the polymer may be hardened in situ. As the composite sets,solvent leaving the composite is preferably released into thesurrounding tissue without causing undesirable side effects such asirritation or an inflammatory response.

The polymer and the particulate phase may be combined by any methodknown to those skilled in the art. For example, a homogenous mixture ofa polymer or polymer precursor and particles may be pressed together atambient or elevated temperatures. At elevated temperatures, the processmay also be accomplished without pressure. In some embodiments, thepolymer or precursor is not held at a temperature of greater thanapproximately 60° C. for a significant time during mixing to preventthermal damage to any biological component of the composite (e.g.,bone-derived factors or cells). Alternatively or in addition, particlesmay be mixed or folded into a polymer softened by heat or a solvent.Alternatively, a formable polymer may be formed into a sheet that isthen covered with a layer of particles. The particles may then be forcedinto the polymer sheet using pressure. In another embodiment, particlesare individually coated with a polymer or polymer precursor, forexample, using a tumbler, spray coater, or a fluidized bed, before beingmixed with a larger quantity of polymer. This facilitates even coatingof the particles and improves integration of the particles and polymercomponent of the composite.

Polymer processing techniques may also be used to combine the particlesand a polymer or polymer precursor. For example, the polymer may berendered formable, e.g., by heating or with a solvent, and combined withthe particles by injection molding or extrusion forming. Alternatively,the polymer and particles may be mixed in a solvent and cast with orwithout pressure. The composite may be prepared from both formable andrigid polymers. For example, extrusion forming may be performed usingpressure to manipulate a formable or rigid polymer.

In another embodiment, the particles may be mixed with a polymerprecursor according to standard composite processing techniques. Forexample, regularly shaped particles may simply be suspended in amonomer. A polymer precursor may be mechanically stirred to distributethe particles or bubbled with a gas, preferably one that is oxygen- andmoisture-free. Once the composite is mixed, it may be desirable to storeit in a container that imparts a static pressure to prevent separationof the particles and the polymer precursor, which may have differentdensities. In some embodiments, the distribution and particle/polymerratio may be optimized to produce at least one continuous path throughthe composite along the particles.

The interaction of the polymer component of the composite with theparticles may also be enhanced by coating individual particles with apolymer precursor before combining them with bulk precursor. The coatingenhances the association of the polymer component of the composite withthe particles. For example, individual particles may be spray coatedwith a monomer or prepolymer. Alternatively, the individual particlesmay be coated using a tumbler—particles and a solid polymer material aretumbled together to coat the particles. A fluidized bed coater may alsobe used to coat the particles. In addition, the particles may simply bedipped into liquid or powdered polymer precursor. All of thesetechniques will be familiar to those skilled in the art.

In some embodiments, it may be desirable to infiltrate a polymer orpolymer precursor into the vascular and/or interstitial structure ofbone-derived particles or into the bone-derived tissue itself. Thevascular structure of bone includes such structures such as osteocytelacunae, Haversian canals, Volksmann's canals, canaliculi and similarstructures. The interstitial structure of the bone particles includesthe spaces between trabeculae and similar features. Many of the monomersand other polymer precursors suggested for use with the invention aresufficiently flowable to penetrate through the channels and pores oftrabecular bone. Some may even penetrate into the trabeculae or into themineralized fibrils of cortical bone. Thus, it may only be necessary toincubate the bone particles in neat monomer or other polymer precursorfor a period of time to accomplish infiltration. In certain embodiments,the polymer itself is sufficiently flowable that it can penetrate thechannels and pores of bone. The polymer may also be heated or combinedwith a solvent to make it more flowable for this purpose. Other ceramicmaterials or bone-substitute materials employed as a particulate phasemay also include porosity that can be infiltrated as described herein.

Vacuum infiltration may be used to help a polymer or precursorinfiltrate the lacunae and canals, and, if desired, the canaliculi.Penetration of the microstructural channels of the bone particles willmaximize the surface area of the interface between the particles and thepolymer and prevent solvents and air bubbles from being trapped in thecomposite, e.g., between trabeculae. Vacuum infiltration, whereappropriate, will also help remove air bubbles from the inventivecomposite.

In another embodiment, infiltration may be achieved using solventinfiltration. Vacuum infiltration may be inappropriate for highlyvolatile monomers. Solvents employed for infiltration should carefullyselected, as many of the most common solvents used for infiltration aretoxic. Highly volatile solvents such as acetone will evaporate duringinfiltration, reducing the risk that they will be incorporated into thepolymer and implanted into the subject. Exemplary solvents for use withthe invention include but are not limited to dimethylsulfoxide (DMSO)and ethanol. As is well known to those skilled in the art, solventinfiltration is achieved by mixing the particles with solutions of thesolvent with the polymer or polymer precursor, starting with very dilutesolutions and proceeding to more concentrated solutions and finally toneat polymer or polymer precursor. Solvent infiltration can also provideimproved tissue infiltration. In some embodiments, solvent infiltrationis combined with pressure in vacuum; instead of finishing theinfiltration with heat monomer, the pressure or vacuum is used to drawout the remaining solvent while pushing the polymer or polymer precursoreven deeper into the particles.

One skilled in the art will recognize that other standard histologicaltechniques, including pressure and heat, may be used to increase theinfiltration of a polymer or polymer precursor into the particles.Infiltrated particles may then be combined with a volume of freshpolymer before administration. Automated apparatus for vacuum andpressure infiltration include the Tissue Tek VIP Vacuum infiltrationprocessor E150/E300, available from Sakura Finetek, Inc.

Alternatively or in addition, a polymer or polymer precursor andparticles may be supplied separately, e.g., in a kit, and mixedimmediately prior to implantation or molding. The kit may contain apreset supply of bone-derived or other particles having, e.g., certainsizes, shapes, and levels of demineralization. The surface of theparticles may have been optionally modified using one or more of thetechniques described herein. Alternatively, the kit may provide severaldifferent types of particles of varying sizes, shapes, and levels ofdemineralization and that may have been chemically modified in differentways. A surgeon or other health care professional may also combine thecomponents in the kit with autologous tissue derived during surgery orbiopsy. For example, the surgeon may want to include autogenous tissueor cells, e.g., bone marrow or bone shavings generated while preparingthe implant site, into the composite.

The composite may include practically any ratio of polymer component andparticles, for example, between about 5 weight % bone and about 95weight % particles. For example, the composite may include about 50% toabout 70% by weight particles. The desired proportion may depend onfactors such as the injection site, the shape and size of the particles,how evenly the polymer is distributed among the particles, desiredflowability of the composite, desired handling of the composite, desiredmoldability of the composite, and the mechanical and degradationproperties of the polymer matrix. The proportions of the polymer andparticles can influence various characteristics of the composite, forexample, its mechanical properties, including fatigue strength, thedegradation rate, and the rate of biological incorporation. In addition,the cellular response to the composite will vary with the proportion ofpolymer and particles. In some embodiments, the desired proportion ofparticles may be determined not only by the desired biologicalproperties of the injected material but by the desired mechanicalproperties of the injected material. That is, an increased proportion ofparticles will increase the viscosity of the composite, making it moredifficult to inject or mold. A larger proportion of particles having awide size distribution may give similar properties to a mixture having asmaller proportion of more evenly sized particles.

One skilled in the art will recognize that standard experimentaltechniques may be used to test these properties for a range ofcompositions to optimize a composite for a desired application. Forexample, standard mechanical testing instruments may be used to test thecompressive strength and stiffness of the composite. Cells may becultured on the composite for an appropriate period of time and themetabolic products and the amount of proliferation (e.g., the number ofcells in comparison to the number of cells seeded) analyzed. The weightchange of the composite may be measured after incubation in saline orother fluids. Repeated analysis will demonstrate whether degradation ofthe composite is linear or not, and mechanical testing of the incubatedmaterial will show the change in mechanical properties as the compositedegrades. Such testing may also be used to compare the enzymatic andnon-enzymatic degradation of the composite and to determine the levelsof enzymatic degradation. A composite that is degraded is transformedinto living bone upon implantation. A non-degradable composite leaves asupporting scaffold which may be interpenetrated with bone or othertissue.

Selection of Polymer

Practically any biocompatible polymer may be used in the composites ofthe invention. Biodegradable polymers may be preferable in someembodiments because composite made from such materials can betransformed into living bone. Polymers that do not degrade may bepreferred for some applications, as they leave a supporting scaffoldthrough which new living tissue may interpenetrate. Co-polymers and/orpolymer blends may also be used in preparing the inventive composites.The selected polymer may be formable and settable under particularconditions, or a monomer or pre-polymer of the polymer may be used. Incertain embodiments, the composite may become more formable when heatedto or over a particular temperature, for example, a temperature at orabove the glass transition temperature or melting point of the polymercomponent. Alternatively, the composite may be more formable when thepolymer component has a certain cross-link density. After the mixture isinjected or molded, the cross-link density of the polymer component ofthe composite may be increased to set the composite. In anotherembodiment, a small amount of monomer is mixed with the polymeric andbone components of the composite. Upon exposure to an energy source,e.g., UV light or heat, the monomer and polymer will further polymerizeand/or cross-link, increasing the molecular weight, the cross-linkdensity, or both. Alternatively or in addition, exposure to body heat, achemical agent, or physiological fluids may stimulate polymerization.

If heat is employed to render the composite and/or the polymer componentof the composite formable, the glass transition or melting temperatureof the polymer component is preferably higher than normal bodytemperature, for example, higher than about 40° C. Polymers that becomemore formable at higher temperatures, e.g., higher than about 45° C.,higher than about 50° C., higher than about 55° C., higher than about60° C., higher than about 70° C., or higher than about 80° C., may alsobe used. However, the temperature required for rendering the compositeformable should not so high as to cause unacceptable tissue necrosisupon implantation. Prior to implantation, the composite is typicallysufficiently cooled to cause little or no tissue necrosis uponimplantation. Exemplary polymers having T_(g) suitable for use with theinvention include but are not limited to starch-poly(caprolactone),poly(caprolactone), poly(lactide), poly(D,L-lactide),poly(lactide-co-glycolide), poly(D,L-lactide-co-glycolide),polycarbonates, polyurethane, tyrosine polycarbonate, tyrosinepolyarylate, poly(orthoesters), polyphosphazenes, polypropylenefumarate, polyhydroxyvalerate, polyhydroxy butyrate, acrylates,methacrylates, and co-polymers, mixtures, enantiomers, and derivativesthereof. In certain particular embodiments, the polymer isstarch-poly(caprolactone), poly(caprolactone), poly(lactide),poly(D,L-lactide), poly(lactide-co-glycolide),poly(D,L-lactide-co-glycolide), polyurethane, or a co-polymer, mixture,enantiomer, or derivative thereof. In certain embodiments, the polymeris poly(D,L-lactide). In certain other embodiments, the polymer ispoly(D,L-lactide-co-glycolide). In certain embodiments, the polymer ispoly(caprolactone). In certain embodiments, the polymer is apoly(urethane). In certain embodiments, the polymer is tyrosinepolycarbonate. In certain embodiments, the polymer is tyrosinepolyarylate.

It is not necessary for all such embodiments that the glass transitiontemperature of the polymer be higher than body temperature. In non-loadbearing and some load-bearing applications, the viscosity of the polymercomponent and resulting composite need only be high enough at bodytemperature that the composite will not flow out of the implant site. Inother embodiments, the polymer component may have crystalline andnon-crystalline regions. Depending on the ratio of crystalline andnon-crystalline material, a polymer may remain relatively rigid betweenthe glass transition and melting temperatures. Indeed, for somepolymers, the melting temperature will determine when the polymermaterial becomes formable.

Since the mixture may be rendered formable just prior to injection,polymer components with glass transition or melting temperatures higherthan 80° C. are also suitable for use with the invention, despite thesensitivity of biological material to heat. For example, PMMA bonecement achieves temperatures of about 50-60° C. during curing. Potentialdamage to bone and/or other materials in the composite depends on boththe temperature and the processing time. As the T_(g) or T_(m) of thepolymer component increases, the composite should be heated for shorterperiods of time to minimize damage to its biological components andshould cool sufficiently quickly to minimize injury at the implantationsite.

The T_(g) of a polymer may be manipulated by adjusting its cross-linkdensity and/or its molecular weight. Thus, for polymers whose glasstransition temperatures are not sufficiently high, increasing thecross-link density or molecular weight can increase the T_(g) to a levelat which composites containing these polymers can be heated to renderthem formable. Alternatively, the polymer may be produced withcrystalline domains, increasing the stiffness of the polymer attemperatures above its glass transition temperature. In addition, theT_(g) of the polymer component may be modified by adjusting thepercentage of the crystalline component. Increasing the volume fractionof the crystalline domains may so reduce the formability of the polymerbetween T_(g) and T_(m) that the composite has to be heated above itsmelting point to be sufficiently formable for use with the invention.

Where a monomer, prepolymer, or other partially polymerized or partiallycross-linked polymer is employed in the inventive composite, theresulting polymer may form by step or chain polymerization. One skilledin the art will recognize that the rate of polymerization should becontrolled so that any change in volume upon polymerization does notimpact mechanical stresses on the included bone particles. The amountand kind of radical initiator, e.g., photo-active initiator (e.g., UV,infrared, or visible), thermally-active initiator, or chemicalinitiator, or the amount of heat or light employed, may be used tocontrol the rate of reaction or modify the molecular weight. Wheredesired, a catalyst may be used to increase the rate of reaction ormodify the molecular weight. For example, a strong acid may be used as acatalyst for step polymerization. Exemplary catalysts for ring openingpolymerization include organotin compounds and glycols and other primaryalcohols. Trifunctional and other multifunctional monomers orcross-linking agents may also be used to increase the cross-linkdensity. For chain polymerizations, the concentration of a chemicalinitiator in the monomer-bone particle mixture may be adjusted tomanipulate the final molecular weight.

Exemplary initiators are listed in George Odian's Principles ofPolymerization, (3rd Edition, 1991, New York, John Wiley and Sons) andavailable from companies such as Polysciences, Wako Specialty Chemicals,Akzo Nobel, and Sigma. Polymerization initiators useful in the inventivecomposite include organic peroxides (e.g., benzoyl peroxide) and azoinitiators (e.g., AIBN). Preferably, the initiator like the polymerand/or monomer is biocompatible. Alternatively, polymerized or partiallypolymerized material may be exposed to UV light, microwaves, or anelectron beam to provide energy for inter-chain reactions.Polymerization may also be triggered by exposure to physiologicaltemperatures or fluids. One skilled in the art will recognize how tomodify the cross-link density to control the rate of degradation and thestiffness of the composite. For example, an accelerator such as anN,N-dialkyl aniline or an N,N-dialkyl toluidine may be used. Exemplarymethods for controlling the rate of polymerization and the molecularweight of the product are also described in Odian (1991), the entirecontents of which are incorporated herein by reference.

Any biocompatible polymer may be used to form composites for use withembodiments of the invention. As noted above, the cross-link density andmolecular weight of the polymer may need to be manipulated so that thepolymer can be formed and set when desired. In some embodiments, theformable polymer material may include monomers, low-molecular weightchains, oligomers, or telechelic chains of the polymers describedherein, and these are cross-linked or further polymerized afterimplantation (e.g., injection). A number of biodegradable andnon-biodegradable biocompatible polymers are known in the field ofpolymeric biomaterials, controlled drug release, and tissue engineering(see, for example, U.S. Pat. Nos. 6,123,727; 5,804,178; 5,770,417;5,736,372; 5,716,404 to Vacanti; U.S. Pat. Nos. 6,095,148; 5,837,752 toShastri; U.S. Pat. No. 5,902,599 to Anseth; U.S. Pat. Nos. 5,696,175;5,514,378; 5,512,600 to Mikos; U.S. Pat. No. 5,399,665 to Barrera; U.S.Pat. No. 5,019,379 to Domb; U.S. Pat. No. 5,010,167 to Ron; U.S. Pat.No. 4,946,929 to d'Amore; and U.S. Pat. Nos. 4,806,621; 4,638,045 toKohn; Beckamn et al., U.S. Patent Application 2005/0013793, publishedJan. 20, 2005; see also Langer, Acc. Chem. Res. 33:94, 2000; Langer, J.Control Release 62:7, 1999; and Uhrich et al., Chem. Rev. 99:3181, 1999,the contents of all of which are incorporated herein by reference).

Other polymers useful in the present invention are described in U.S.patent applications, U.S. Ser. No. 10/735,135, filed on Dec. 12, 2003,entitled “Formable and settable polymer bone composite and method ofproduction thereof” and published under No. 2005-0008672; U.S. Ser. No.10/681,651, filed on Oct. 8, 2003, entitled “Coupling agents fororthopedic biomaterials” and published under No. 2005-0008620; and U.S.Ser. No. 60/760,538, filed on Jan. 19, 2006 and entitled “Injectable andSettable Bone Substitute Material”, all of which are incorporated hereinby reference.

In one embodiment, the polymer matrix is biodegradable. Exemplarybiodegradable materials include lactide-glycolide copolymers of anyratio (e.g., 85:15, 40:60, 30:70, 25:75, or 20:80),poly(L-lactide-co-D,L-lactide), polyglyconate, poly(arylates),poly(anhydrides), poly(hydroxy acids), polyesters, poly(ortho esters),poly(alkylene oxides), polycarbonates, poly(propylene fumarates),poly(propylene glycol-co fumaric acid), poly(caprolactones), polyamides,polyesters, polyethers, polyureas, polyamines, polyamino acids,polyacetals, poly(orthoesters), poly(pyrolic acid), poly(glaxanone),poly(phosphazenes), poly(organophosphazene), polylactides,polyglycolides, poly(dioxanones), polyhydroxybutyrate,polyhydroxyvalyrate, polyhydroxybutyrate/valerate copolymers, poly(vinylpyrrolidone), biodegradable polycyanoacrylates, biodegradablepolyurethanes including glucose-based polyurethanes and lysine-basedpolyurethanes, and polysaccharides (e.g., chitin, starches, celluloses).In certain embodiments, the polymer used in the inventive composite ispoly(lactide-co-glycolide). The ratio of lactide and glycolide units inthe polymer may vary. Particularly useful ratios are approximately45-80% lactide to approximately 44-20% glycolide. In certainembodiments, the ratio is approximately 50% lactide to approximately 50%glycolide. In other certain embodiments, the ratio is approximately 65%lactide to approximately 45% glycolide. In other certain embodiments,the ratio is approximately 60% lactide to approximately 40% glycolide.In other certain embodiments, the ratio is approximately 70% lactide toapproximately 30% glycolide. In other certain embodiments, the ratio isapproximately 75% lactide to approximately 25% glycolide. In certainembodiments, the ratio is approximately 80% lactide to approximately 20%glycolide. In certain of the above embodiments, lactide is D,L-lactide.In other embodiments, lactide is L-lactide. In certain particularembodiments, RESOMER® 824 (poly-L-lactide-co-glycolide) (BoehringerIngelheim) is used as the polymer in the composite. In certainparticular embodiments, RESOMER® 504 (poly-D,L-lactide-co-glycolide)(Boehringer Ingelheim) is used as the polymer in the composite. Incertain particular embodiments, PURASORB PLG (75/25poly-L-lactide-co-glycolide) (Purac Biochem) is used as the polymer inthe composite. In certain particular embodiments, PURASORB PG(polyglycolide) (Purac Biochem) is used as the polymer in the composite.In certain embodiments, the polymer isPEGylated-poly(lactide-co-glycolide). In certain embodiments, thepolymer is PEGylated-poly(lactide). In certain embodiments, the polymeris PEGylated-poly(glycolide). In other embodiments, the polymer ispolyurethane. In other embodiments, the polymer is polycaprolactone. Incertain embodiments, the polymer is a co polymer of poly(caprolactone)and poly(lactide). For polyesters such as poly(lactide) andpoly(lactide-co-glycolide), the inherent viscosity of the polymer rangesfrom about 0.4 dL/g to about 5 dL/g. In certain embodiments, theinherent viscosity of the polymer ranges from about 0.6 dL/g to about 2dL/g. In certain embodiments, the inherent viscosity of the polymerranges from about 0.6 dL/g to about 3 dL/g. In certain embodiments, theinherent viscosity of the polymer ranges from about 1 dL/g to about 3dL/g. In certain embodiments, the inherent viscosity of the polymerranges from about 0.4 dL/g to about 1 dL/g. For poly(caprolactone), theinherent viscosity of the polymer ranges from about 0.5 dL/g to about1.5 dL/g. In certain embodiments, the inherent viscosity of thepoly(caprolactone) ranges from about 1.0 dL/g to about 1.5 dL/g. Incertain embodiments, the inherent viscosity of the poly(caprolactone)ranges from about 1.0 dL/g to about 1.2 dL/g. In certain embodiments,the inherent viscosity of the poly(caprolactone) is about 1.08 dL/g.Natural polymers, including collagen, polysaccharides, agarose,glycosaminoglycans, alginate, chitin, and chitosan, may also beemployed. Tyrosine-based polymers, including but not limited topolyarylates and polycarbonates, may also be employed (see Pulapura, etal., “Tyrosine-derived polycarbonates: Backbone-modified“pseudo”-poly(amino acids) designed for biomedical applications,”Biopolymers, 1992, 32: 411-417; Hooper, et al., “Diphenolic monomersderived from the natural amino acid α-L-tyrosine: an evaluation ofpeptide coupling techniques,” J. Bioactive and Compatible Polymers,1995, 10:327-340, the contents of both of which are incorporated hereinby reference). Monomers for tyrosine-based polymers may be prepared byreacting an L-tyrosine-derived diphenol compound with phosgene or adiacid (Hooper, 1995; Pulapura, 1992). Similar techniques may be used toprepare amino acid-based monomers of other amino acids having reactiveside chains, including imines, amines, thiols, etc. The polymersdescribed in the application entitled “Polyurethanes for Osteoimplants,”filed on even date herewith, may also be used in embodiments of thepresent invention. In one embodiment, the degradation products includebioactive materials, biomolecules, small molecules, or other suchmaterials that participate in metabolic processes.

Polymers may be manipulated to adjust their degradation rates. Thedegradation rates of polymers are well characterized in the literature(see Handbook of Biodegradable Polymers, Domb, et al., eds., HarwoodAcademic Publishers, 1997, the entire contents of which are incorporatedherein by reference). In addition, increasing the cross-link density ofa polymer tends to decrease its degradation rate. The cross-link densityof a polymer may be manipulated during polymerization by adding across-linking agent or promoter. After polymerization, cross-linking maybe increased by exposure to UV light or other radiation. Co-monomers ormixtures of polymers, for example, lactide and glycolide polymers, maybe employed to manipulate both degradation rate and mechanicalproperties.

Non-biodegradable polymers may also be used as well. For example,polypyrrole, polyanilines, polythiophene, and derivatives thereof areuseful electroactive polymers that can transmit voltage from endogenousbone to an implant. Other non-biodegradable, yet biocompatible polymersinclude polystyrene, polyesters, polyureas, poly(vinyl alcohol),polyamides, poly(tetrafluoroethylene), and expandedpolytetrafluroethylene (ePTFE), poly(ethylene vinyl acetate),polypropylene, polyacrylate, non-biodegradable polycyanoacrylates,non-biodegradable polyurethanes, mixtures and copolymers of poly(ethylmethacrylate) with tetrahydrofurfuryl methacrylate, polymethacrylate,poly(methyl methacrylate), polyethylene, including ultra high molecularweight polyethylene (UHMWPE), polypyrrole, polyanilines, polythiophene,poly(ethylene oxide), poly(ethylene oxide co-butylene terephthalate),poly ether-ether ketones (PEEK), and polyetherketoneketones (PEKK).Monomers that are used to produce any of these polymers are easilypurchased from companies such as Polysciences, Sigma, and ScientificPolymer Products.

Those skilled in the art will recognize that this is an exemplary, not acomprehensive, list of polymers appropriate for in vivo applications.Co-polymers, mixtures, and adducts of the above polymers may also beused with the invention.

Additional Components

Additional materials may be included in the inventive composite. Theadditional material may be biologically active or inert. Additionalmaterials may also be added to the composite to improve its chemical,mechanical, or biophysical properties. Additional materials may also beadded to improve the handling or storage of the composite (e.g., apreservative). Those of skill in this art will appreciate the myriad ofdifferent components that may be included in the composite.

Additional components of the composite may be any type of chemicalcompound including proteins, peptides, polynucleotides (e.g., vectors,plasmids, cosmids, artificial chromosomes, etc.), lipids, carbohydrates,organic molecules, small molecules, organometallic compounds, metals,ceramics, polymers, etc. Living cells, tissue samples, or viruses mayalso be added to the inventive composites. In certain embodiments, theadditional material comprises cells, which may optionally be geneticallyengineered. For example, the cells may be engineered to produce aspecific growth factor, chemotactic factor, osteogenic factor, etc. Incertain embodiments, the cells may be engineered to produce apolynucleotide such as an siRNA, shRNA, RNAi, microRNA, etc. The cellmay include a plasmid, or other extra-chromosomal piece of DNA. Incertain embodiments, a recombinant construct is integrated into thegenome of the cell. In certain embodiments, the additional materialcomprises a virus. Again, the virus may be genetically engineered.Tissues such as bone marrow and bone samples may be combined with thecomposite of polymer and bone-derived particles. The composite mayinclude additional calcium-based ceramics such as calcium phosphate andcalcium carbonate. In certain embodiments, non-biologically activematerials are incorporated into the composite. For example, labelingagents such as radiopaque, luminescent, or magnetically active particlesmay be attached to the bone-derived particles using silane chemistry orother coupling agents, for example zirconates and titanates, or mixedinto the polymer, as described herein. Alternatively, or in addition,poly(ethylene glycol) (PEG) may be attached to the bone particles.Biologically active molecules, for example, small molecules, bioactiveagents, and biomolecules such as lipids may be linked to the particlesthrough silane SAMs or using a polysialic acid linker (see, for example,U.S. Pat. No. 5,846,951; incorporated herein by reference).

The composite may also include one or more other components such as aplasticizer. Plasticizer are typically compounds added to polymers orplastics to soften them or make them more pliable. Plasticizers soften,make workable, or otherwise improve the handling properties of a polymeror composite. Plasticizers also allow the inventive composite to bemoldable at a lower temperature, thereby avoiding heat induced tissuenecrosis during implantation. The plasticizer may evaporate or otherwisediffuse out of the composite over time, thereby allowing the compositeto harden or set. Plasticizer are thought to work by embeddingthemselves between the chains of polymers. This forces the polymerchains apart and thus lowers the glass transition temperature of thepolymer. Typically, the more plasticizer that is added, the moreflexible the resulting polymer or composite will be.

In certain embodiments, the plasticizer is based on an ester of apolycarboxylic acid with linear or branched aliphatic alcohols ofmoderate chain length. For example, some plasticizers are adipate-based.Examples of adipate-based pasticizers include bis(2-ethylhexyl)adipate(DOA), dimethyl adipate (DMAD), monomethyl adipate (MMAD), and dioctyladipate (DOA). Other plasticizers are based on maleates, sebacates, orcitrates such as bibutyl maleate (DBM), diisobutylmaleate (DIBM),dibutyl sebacate (DBS), triethyl citrate (TEC), acetyl triethyl citrate(ATEC), tributyl citrate (TBC), acetyl tributyl citrate (ATBC), trioctylcitrate (TOC), acetyl trioctyl citrate (ATOC), trihexyl citrate (THC),acetyl trihexyl citrate (ATHC), butyryl trihexyl citrate (BTHC), andtrimehtylcitrate (TMC). Other plasticizers are phthalate based. Examplesof phthalate-based plasticizers are N-methyl phthalate,bis(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP),bis(n-butyl)phthalate (DBP), butyl benzyl phthalate (BBzP), diisodecylphthalate (DOP), diethyl phthalate (DEP), diisobutyl phthalate (DIBP),and di-n-hexyl phthalate. Other suitable plasticizers include liquidpolyhydroxy compounds such as glycerol, polyethylene glycol (PEG),triethylene glycol, sorbitol, monacetin, diacetin, and mixtures thereof.Other plasticizers include trimellitates (e.g., trimethyl trimellitate(TMTM), tri-(2-ethylhexyl) trimellitate (TEHTM-MG),tri-(n-octyl,n-decyl) trimellitate (ATM), tri-(heptyl,nonyl)trimellitate (LTM), n-octyl trimellitate (OTM)), benzoates, epoxidizedvegetable oils, sulfonamides (e.g., N-ethyl toluene sulfonamide (ETSA),N-(2-hydroxypropyl) benzene sulfonamide (HP BSA), N-(n-butyl) butylsulfonamide (BBSA-NBBS)), organophosphates (e.g., tricresyl phosphate(TCP), tributyl phosphate (TBP)), glycols/polyethers (e.g., triethyleneglycol dihexanoate, tetraethylene glycol diheptanoate), and polymericplasticizers. Other plasticizers are described in Handbook ofPlasticizers (G. Wypych, Ed., ChemTec Publishing, 2004), which isincorporated herein by reference. In certain embodiments, other polymersare added to the composite as plasticizers. In certain particularembodiments, polymers with the same chemical structure as those used inthe composite are used but with lower molecular weights to soften theoverall composite. In certain embodiments, oligomers or monomers of thepolymers used in the composite are used as plasticizers. In otherembodiments, different polymers with lower melting points and/or lowerviscosities than those of the polymer component of the composite areused. In certain embodiments, oligomers or monomers of polymersdifferent from those used in the composite are used as plasticizers. Incertain embodiments, the polymer used as a plasticizer is poly(ethyleneglycol) (PEG). The PEG used as a plasticizer is typically a lowmolecular weight PEG such as those having an average molecular weight of1000 to 10000 g/mol, preferably from 4000 to 8000 g/mol. In certainembodiments, PEG 4000 is used in the composite. In certain embodiments,PEG 5000 is used in the composite. In certain embodiments, PEG 6000 isused in the composite. In certain embodiments, PEG 7000 is used in thecomposite. In certain embodiments, PEG 8000 is used in the composite.The plasticizer (PEG) is particularly useful in making more moldablecomposites that include poly(lactide), poly(D,L-lactide),poly(lactide-co-glycolide), poly(D,L-lactide-co-glycolide), orpoly(caprolactone). In certain embodiments, PEG is grafted onto apolymer of the composite or is co-polymerized with a polymer of thecomposite.

Plasticizer may comprise 1-40% of the composite by weight. In certainembodiments, the plasticizer is 10-30% by weight. In certainembodiments, the plasticizer is approximately 10% by weight. In certainembodiments, the plasticizer is approximately 15% by weight. In certainembodiments, the plasticizer is approximately 20% by weight. In certainembodiments, the plasticizer is approximately 25% by weight. In certainembodiments, the plasticizer is approximately 30% by weight. In certainembodiments, the plasticizer is approximately 33% by weight. In certainembodiments, the plasticizer is approximately 40% by weight. In certainembodiments, a plasticizer is not used in the composite. For example, insome polycaprolactone-containing composites, a plasticizer is not used.

The inventive composite may be porous (e.g., at the time ofmanufacture), may be made porous prior to implantation, or may becomeporous upon implantation. For a general discussion of the use ofporosity in osteoimplants, see U.S. patent application US 2005/0251267,published Nov. 10, 2005; which is incorporated herein by reference. Aporous composite osteoimplant with an interconnecting network of poreshas been shown to facilitate the invasion of cells and promote theorganized growth of incoming cells and tissue (e.g., living bone).Allcock et al. “Synthesis of poly[(amino acid alkyl ester) phosphazenes”Macromolecules 10:824-830, 1977; Allcock et al. “Hydrolysis pathways foraminophosphazenes” Inorg. Chem. 21:515-521, 1982; Mikos et al.“Prevascularization of biodegradable polymer scaffolds for hepatocytetransplantation” Proc. ACS Div. of Polymer Mater. 66:33, 1992; Eggli etal. “Porous hydroxyapatite and tricalcium phosphate cylinders with twodifferent pore size ranges implanted in the cancellous bone of rabbits”Clin. Orthop. 232:127-138, 1987; each of which is incorporated herein byreference. Porosity has also been shown to influence thebiocompatibility and bony integration of polymeric composites. White etal. “Biomaterial aspects of Interpore 200 porous hydroxyapatite” DentalClinical of N. Amer. 30:49-67, 1986; which is incorporated herein byreference.

The porosity of the composite may include both open and closed cells.The terms “open cells” and “open-celled structure” are used hereininterchangeably and refer to a porous material with very largepermeability, and where no significant surface barriers exist betweencells (i.e., where the pores are connected). The terms “closed cells”and “close-celled structure” are used herein interchangeably and referto a porous material where the pores are not connected, resulting in aweakly permeable material. Open cells in an inventive composite increasethe paths for tissue to infiltrate the composite and will decreasedegradation times. The proportion and size distribution ranges of openand closed cells of the final inventive composite (e.g., before or afterimplantation) may be adjusted by controlling such factors as theidentity of the porogen, percentage of porogen, percentage of particles,the properties of the polymer, etc.

The composites of the present invention can exhibit high degrees ofporosity over a wide range of effective pore sizes. Thus, composites ofthe present invention may have, at once, macroporosity, mesoporosity andmicroporosity. Macroporosity is characterized by pore diameters greaterthan about 100 microns. Mesoporosity is characterized by pore diametersbetween about 100 microns about 10 microns; and microporosity occurswhen pores have diameters below about 10 microns. In some embodiments,the composite has a porosity of at least about 30%. For example, incertain embodiments, the composite has a porosity of more than about50%, more than about 60%, more than about 70%, more than bout 80%, ormore than about 90%. Advantages of a highly porous composite over lessporous or non-porous composite include, but are not limited to, moreextensive cellular and tissue in-growth into the composite, morecontinuous supply of nutrients, more thorough infiltration oftherapeutics, and enhanced revascularization, allowing bone growth andrepair to take place more efficiently. Furthermore, in certainembodiments, the porosity of the composite may be used to load thecomposite with biologically active agents such as drugs, smallmolecules, cells, peptides, polynucleotides, growth factors, osteogenicfactors, etc, for delivery at the implant site. Porosity may also rendercertain composites of the present invention compressible.

In certain particular embodiments, the pores of the composite arepreferably over 100 microns wide for the invasion of cells and bonyin-growth. Klaitwatter et al. “Application of porous ceramics for theattachment of load bering orthopedic applications” J. Biomed. Mater.Res. Symp. 2:161, 1971; each of which is incorporated herein byreference. In certain embodiments, the pore size ranges fromapproximately 50 microns to approximately 500 microns, preferably fromapproximately 100 microns to approximately 250 microns.

The porosity of the composite may be accomplished using any means knownin the art. Exemplary methods of creating porosity in a compositeinclude, but are not limited to, particular leaching processes, gasfoaming processing, supercritical carbon dioxide processing, sintering,phase transformation, freeze-drying, cross-linking, molding, porogenmelting, polymerization, melt-blowing, and salt fusion (Murphy et al.Tissue Engineering 8(1):43-52, 2002; incorporated herein by reference).For a review, see Karageorgiou et al., Biomaterials 26:5474-5491, 2005;incorporated herein by reference. The porosity may be a feature of thecomposite during manufacture or before implantation, or the porosity mayonly be available after implantation. For example, the implantedcomposite may include latent pores. These latent pores may arise fromincluding porogens in the composite.

The porogen may be any chemical compound that will reserve a spacewithin the composite while the composite is being molded and willdiffuse, dissolve, and/or degrade prior to or after implantation leavinga pore in the composite. Porogens preferably have the property of notbeing appreciably changed in shape and/or size during the procedure tomake the composite moldable. For example, the porogen should retain itsshape during the heating of the composite to make it moldable.Therefore, the porogen preferably does not melt upon heating of thecomposite to make it moldable. In certain embodiments, the porogen has amelting point greater than about 60° C., greater than about 70° C.,greater than about 80° C., greater than about 85° C., or greater thanabout 90° C.

Porogens may be of any shape or size. The porogen may be spheroidal,cuboidal, rectangular, elongated, tubular, fibrous, disc-shaped,platelet-shaped, polygonal, etc. In certain embodiments, the porogen isgranular with a diameter ranging from approximately 100 microns toapproximately 800 microns. In certain embodiments, the porogen iselongated, tubular, or fibrous. Such porogens provide increasedconnectivity of the pores of the composite and/or also allow for alesser percentage of the porogen in the composite. The amount of theporogen may vary in the composite from 1% to 80% by weight. In certainembodiments, the plasticizer makes up from about 5% to about 80% byweight of the composite. In certain embodiments, the plasticizer makesup from about 10% to about 50% by weight of the composite. Pores in thecomposite are thought to improve the osteoinductivity orosteoconductivity of the composite by providing holes for cells such asosteoblasts, osteoclasts, fibroblasts, cells of the osteoblast lineage,stem cells, etc. The pores provide the composite with biological ingrowth capacity. Pores in the composite may also provide for easierdegradation of the composite as bone is formed and/or remodeled.Preferably, the porogen is biocompatible.

The porogen may be a gas, liquid, or solid. Exemplary gases that may actas porogens include carbon dioxide, nitrogen, argon, or air. Exemplaryliquids include water, organic solvents, or biological fluids (e.g.,blood, lymph, plasma). The gaseous or liquid porogen may diffuse out ofthe osteoimplant before or after implantation thereby providing poresfor biological in-growth. Solid porogens may be crystalline oramorphous. Examples of possible solid porogens include water solublecompounds. In certain embodiments, the water soluble compound has asolubility of greater than 10 g per 100 mL water at 25° C. In certainembodiments, the water soluble compound has a solubility of greater than25 g per 100 mL water at 25° C. In certain embodiments, the watersoluble compound has a solubility of greater than 50 g per 100 mL waterat 25° C. In certain embodiments, the water soluble compound has asolubility of greater than 75 g per 100 mL water at 25° C. In certainembodiments, the water soluble compound has a solubility of greater than100 g per 100 mL water at 25° C. Examples of porogens includecarbohydrates (e.g., sorbitol, dextran (poly(dextrose)), starch), salts,sugar alcohols, natural polymers, synthetic polymers, and smallmolecules.

In certain embodiments, carbohydrates are used as porogens in theinventive composites. The carbohydrate may be a monosaccharide,disaccharide, or polysaccharide. The carbohydrate may be a natural orsynthetic carbohydrate. Preferably, the carbohydrate is a biocompatible,biodegradable carbohydrate. In certain embodiments, the carbohydrate isa polysaccharide. Exemplary polysaccharides include cellulose, starch,amylose, dextran, poly(dextrose), glycogen, etc. In certain embodiments,the polysaccharide is dextran. Very high molecular weight dextran hasbeen found particularly useful as a porogen. For example, the molecularweight of the dextran may range from about 500,000 g/mol to about10,000,000 g/mol, preferably from about 1,000,000 g/mol to about3,000,000 g/mol. In certain embodiments, the dextran has a molecularweight of approximately 2,000,000 g/mol. Dextrans with a molecularweight higher than 10,000,000 g/mol may also be used as porogens.Dextran may be used in any form (e.g., particles, granules, fibers,elongated fibers) as a porogen. In certain embodiments, fibers orelongated fibers of dextran are used as the porogen in the inventivecomposite. Fibers of dextran may be formed using any known methodincluding extrusion and precipitation. Fibers may be prepared byprecipitation by adding an aqueous solution of dextran (e.g., 5-25%dextran) to a less polar solvent such as a 90-100% alcohol (e.g.,ethanol) solution. The dextran precipitates out in fibers that areparticularly useful as porogens in the inventive composite. Dextran maybe about 15% by weight to about 30% by weight of the composite. Incertain embodiments, dextran is about 15% by weight, 20% by weight, 25%by weight, or 30% by weight. Higher and lower percentages of dextran mayalso be used. Once the composite with the dextran as a porogen isimplanted into a subject, the dextran dissolves away very quickly.Within approximately 24 hours, substantially all of the dextran is outof the composite leaving behind pores in the osteoimplant composite. Anadvantage of using dextran in the composite is that dextran exhibits ahemostatic property in the extravascular space. Therefore, dextran in acomposite can decrease bleeding at or near the site of implantation.

Small molecules including pharmaceutical agents may also be used asporogens in the inventive composites. Examples of polymers that may beused as plasticizers include poly(vinyl pyrollidone), pullulan,poly(glycolide), poly(lactide), and poly(lactide-co-glycolide).Typically low molecular weight polymers are used as porogens. In certainembodiments, the porogen is poly(vinyl pyrrolidone) or a derivativethereof. Plasticizers that are removed faster than the surroundingcomposite can also be considered porogens.

In certain embodiments, the composite may include a wetting orlubricating agent. Suitable wetting agents include water, organic proticsolvents, organic non-protic solvents, aqueous solutions such asphysiological saline, concentrated saline solutions, sugar solutions,ionic solutions of any kind, and liquid polyhydroxy compounds such asglycerol, polyethylene glycol (PEG), polyvinyl alcohol (PVA), andglycerol esters, and mixtures of any of these. Biological fluids mayalso be used as wetting or lubricating agents. Examples of biologicalfluids that may be used with the inventive composites include blood,lymph, plasma, serum, or marrow. Lubricating agents may include, forexample, polyethylene glycol, which can be combined with the polymer andother components to reduce viscosity or even coated on the walls of thedelivery device. Alternatively or in addition, the particulate materialmay be coated with a polymer by sputtering or other techniques known tothose skilled in the art.

Additionally, composites of the present invention may contain one ormore biologically active molecules, including biomolecules, smallmolecules, and bioactive agents, to promote bone growth and connectivetissue regeneration, and/or to accelerate healing. Examples of materialsthat can be incorporated include chemotactic factors, angiogenicfactors, bone cell inducers and stimulators, including the general classof cytokines such as the TGF-β superfamily of bone growth factors, thefamily of bone morphogenic proteins, osteoinductors, and/or bone marrowor bone forming precursor cells, isolated using standard techniques.Sources and amounts of such materials that can be included are known tothose skilled in the art.

In certain embodiments, the composite include antibiotics. Theantibiotics may be bacteriocidial or bacteriostatic. Otheranti-microbial agents may also be included in the composite. Forexample, anti-viral agents, anti-protazoal agents, anti-parasiticagents, etc. may be include in the composite. Other suitablebiostatic/biocidal agents include antibiotics, povidone, sugars, andmixtures thereof.

Biologically active materials, including biomolecules, small molecules,and bioactive agents may also be combined with the polymer and particlesto, for example, stimulate particular metabolic functions, recruitcells, or reduce inflammation. For example, nucleic acid vectors,including plasmids and viral vectors, that will be introduced into thepatient's cells and cause the production of growth factors such as bonemorphogenetic proteins may be included in the composite. Biologicallyactive agents include, but are not limited to, antiviral agent,antimicrobial agent, antibiotic agent, amino acid, peptide, protein,glycoprotein, lipoprotein, antibody, steroidal compound, antibiotic,antimycotic, cytokine, vitamin, carbohydrate, lipid, extracellularmatrix, extracellular matrix component, chemotherapeutic agent,cytotoxic agent, growth factor, anti-rejection agent, analgesic,anti-inflammatory agent, viral vector, protein synthesis co-factor,hormone, endocrine tissue, synthesizer, enzyme, polymer-cell scaffoldingagent with parenchymal cells, angiogenic drug, collagen lattice,antigenic agent, cytoskeletal agent, mesenchymal stem cells, bonedigester, antitumor agent, cellular attractant, fibronectin, growthhormone cellular attachment agent, immunosuppressant, nucleic acid,surface active agent, hydroxyapatite, and penetraction enhancer.Additional exemplary substances include chemotactic factors, angiogenicfactors, analgesics, antibiotics, anti-inflammatory agents, bonemorphogenic proteins, and other growth factors that promotecell-directed degradation or remodeling of the polymer phase of thecomposite and/or development of new tissue (e.g., bone). RNAi or othertechnologies may also be used to reduce the production of variousfactors.

To enhance biodegradation in vivo, the composites of the presentinvention can also include different enzymes. Examples of suitableenzymes or similar reagents are proteases or hydrolases withester-hydrolyzing capabilities. Such enzymes include, but are notlimited to, proteinase K, bromelaine, pronase E, cellulase, dextranase,elastase, plasmin streptokinase, trypsin, chymotrypsin, papain,chymopapain, collagenase, subtilisin, chlostridopeptidase A, ficin,carboxypeptidase A, pectinase, pectinesterase, an oxireductase, anoxidase, or the like. The inclusion of an appropriate amount of such adegradation enhancing agent can be used to regulate implant duration.

These materials need not be covalently bonded to a component of thecomposite. A material may be selectively distributed on or near thesurface of the composite using the layering techniques described above.While the surface of the composite will be mixed somewhat as thecomposite is manipulated in the implant site, the thickness of thesurface layer will ensure that at least a portion of the surface layerof the composite remains at the surface of the implant. Alternatively orin addition, biologically active components may be covalently linked tothe bone particles before combination with the polymer. For example,silane coupling agents having amine, carboxyl, hydroxyl, or mercaptogroups may be attached to the bone particles through the silane and thento reactive groups on a biomolecule, small molecule, or bioactive agent.

The composite may also be seeded with cells. In certain embodiments, apatient's own cells are obtained and used in the inventive composite.Certain types of cells (e.g., osteoblasts, fibroblasts, stem cells,cells of the osteoblast lineage, etc.) may be selected for use in thecomposite. The cells may be harvested from marrow, blood, fat, bone,muscle, connective tissue, skin, or other tissues or organs. In certainembodiments, a patient's own cells may be harvested, optionallyselected, expanded, and used in the inventive composite. In otherembodiments, a patient's cells may be harvested, selected withoutexpansion, and used in the inventive composite. Alternatively, exogenouscells may be employed. Exemplary cells for use with the inventioninclude mesenchymal stem cells and connective tissue cells, includingosteoblasts, osteoclasts, fibroblasts, preosteoblasts, and partiallydifferentiated cells of the osteoblast lineage. The cells may begenetically engineered. For example, the cells may be engineered toproduce a bone morphogenic protein.

In embodiments where the polymer component becomes formable when heated,the heat absorbed by particles in the composite may increase the coolingtime of the composite, extending the time available to form thecomposite into an implant. Depending on the relative heat capacities ofthe particle and the polymer components and the size of the particles,the particles may continue to release heat into the surrounding polymerafter the time when the polymer alone would have cooled. The size anddensity distribution of particles within the composite may be optimizedto adjust the amount of heat released into portions of an osteoimplantduring and after implantation.

Administration of the Composite Material

The inventive composite may be administered to a subject in need thereofusing any technique known in the art. The subject is typically a patientwith a disorder or disease related to bone. In certain embodiments, thesubject has a bony defect such as a fracture. The subject is typically amammal although any animal with bones may benefit from treatment withthe inventive composite. In certain embodiments, the subject is avertebrate (e.g., mammals, reptiles, fish, birds, etc.). In certainembodiments, the subject is a human. In other embodiments, the subjectis a domesticated animal such as a dog, cat, horse, etc. Any bonedisease or disorder may be treated using the inventive compositeincluding genetic diseases, congenital abnormalities, fractures,iatrogenic defects, bone cancer, bone metastases, inflammatory diseases(e.g. rheumatoid arthritis), autoimmune diseases, metabolic diseases,and degenerative bone disease (e.g., osteoarthritis). In certainembodiments, the inventive osteoimplant composites are formulated forthe repair of a simple fracture, compound fracture, or non-union; as anexternal fixation device or internal fixation device; for jointreconstruction, arthrodesis, arthroplasty, or cup arthroplasty of thehip; for femoral or humeral head replacement; for femoral head surfacereplacement or total joint replacement; for repair of the vertebralcolumn, spinal fusion or internal vertebral fixation; for tumor surgery;for deficit filling; for discectomy; for laminectomy; for excision ofspinal tumors; for an anterior cervical or thoracic operation; for therepairs of a spinal injury; for scoliosis, for lordosis or kyphosistreatment; for intermaxillary fixation of a fracture; for mentoplasty;for temporomandibular joint replacement; for alveolar ridge augmentationand reconstruction; as an inlay osteoimplant; for implant placement andrevision; for sinus lift; for a cosmetic procedure; for revisionsurgery; for revision surgery of a total joint arthroplasty; and for therepair or replacement of the ethmoid, frontal, nasal, occipital,parietal, temporal, mandible, maxilla, zygomatic, cervical vertebra,thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle,scapula, humerus, radius, ulna, carpal bones, metacarpal bones,phalanges, ilium, ischium, pubis, femur, tibia, fibula, patella,calcaneus, tarsal bones, or metatarsal bones. In certain embodiments,the inventive composite is used to seal a defect, void, or hole in abone. For example, a bony defect may be filled with mineralized and/orpartially or fully demineralized allograft bone or other bone substitutematerial, and the defect is sealed with the inventive composite.

The composite material is typically administered to a patient in aclinical setting. In certain embodiments, the osteoimplant composite isadministered during a surgical procedure. The osteoimplant composite maybe placed at an implant site by molding, placing, injecting, orextruding the inventive composite into the site of implantation. Thecomposite is typically made moldable or flowable before it isadministered to a subject. This allows the composite to fit intoirregularly shaped sites. In certain embodiments, the composite of theparticles with the polymer is injected or extruded into a tissue site(e.g., a bony defect). In one embodiment, the mixture is injected usingminimally invasive surgical techniques or through a transcutaneousprocedure such as percutaneous vertebroplasty. The procedure may notrequire a surgical incision or opening of the patient as required fortraditional surgical procedures. For example, the mixture may beinjected using a needle and syringe. The syringe may be driven by handor mechanically. The needle may be positioned by radiological meansbefore injection of the composite. It may be desirable to include arigid injection system to provide more precise control over the injectedvolume.

The technique employed to deliver the flowable composite depends in parton the flow rate F of the material through the delivery device, which inturn depends in part on the resistance to flow of the composite. Forlaminar flow, the resistance to flow R is defined by the Poisseuleequation,

$\begin{matrix}{R = \frac{8\eta\; L}{\pi\; r^{4}}} & (1)\end{matrix}$where η is the viscosity, L is the length of the flow, and r is theradius of the bore through which the material is flowing. Thus, forinjection through a long needle or deep into a tissue, a larger borecannula may be useful to reduce flow resistance. Back pressure from theinjection site may also dictate the desired cannula size or deliverydevice, since the flow rate depends on the back pressure as

$\begin{matrix}{F = \frac{P_{1} - P_{2}}{R}} & (2)\end{matrix}$where P₁ and P₂ are the inlet and outlet pressures of the cannula.

One skilled in the art will recognize that one of the factorsinfluencing the length of the flow is the distance from the injectionsite to the access point for the extruder or needle. In someembodiments, the mixture is injected percutaneously. A bony injectionsite may be some distance from the skin, necessitating a longer needle.In other embodiments, the injection site may be exposed, for example,during surgery. In these cases a very short cannula may suffice fordelivery of the mixture, and a wider bore cannula may be appropriate.

One skilled in the art will recognize that a variety of cannula sizesmay be employed to deliver mixtures according to embodiments of theinvention. For example, a wider gauge may be desired for longercannulae. Depending on the factors below, cannulae of 6 gauge ornarrower, for example, 7 gauge, 8 gauge, 9 gauge, 10 gauge, 11 gauge, or12 gauge, may be employed for percutaneous injection. In certainparticular embodiments, a cannulae of 10 gauge, 11 gauge, or 12 gauge isused. Where the injection site is exposed or the injection is made usingminimally invasive surgical techniques, even wider cannulae, e.g., 5gauge, 3 gauge, about 1 cm, or wider. The optimal wall thickness may beeasily tested by testing the yield strength of the needles underpressure. The taper on needles and cannulae may be optimized for thetissue or material that needs to be penetrated, independently of thecharacteristics of the composite being delivered.

The flow characteristics of the composite are also influenced by theratio of the carrier to the solid particles and the interaction of theparticles with the carrier and with each other. As the composite isinjected, a “filter cake” may form at the entrance to the cannula;likewise, porous tissue at the implant site may also act as a filter,allowing the carrier to flow more easily than the particles andpromoting the formation of a filter cake. Filtering may be alleviated byincreasing the difference between the actual carrier/particle ratio andthe plastic limit. The plastic limit may be decreased by using moreregularly shaped particles instead of elongated particles, by increasingthe breadth of the particle size distribution, by reducing agglomerationof the dry particles before blending with the carrier, and by reducingthe degree of interparticle interactions, for example by changing thesurface charge or by adsorbing a polymer onto the surface of theparticles. The thickness of the filter cake is directly proportional tothe particle size, proportional to the square root of the cannulalength, and inversely proportional to the square of the internaldiameter of the cannula. Decreasing the delivery rate may also reducefiltering.

Another factor influencing the delivery is the potential degree ofextravasation, the “leaking” of the mixture into the marrow space oroutside the bone tissue, e.g., when the composite is injected into bonetissue. Extravasation may be reduced by increasing the viscosity of thecomposite. In many cases, extravasation may be prevented or reduced ifthe pressure required for extravasation is greater than that required toinject the composite into the desired site, which need not be the sameas P₂. However, as the porosity and the pore size at the injection sitedecrease, the pressure required to infiltrate the tissue increases.

In other embodiments, the composite is molded into a shape that can beplaced into a tissue site. After placement, the composite may be furthermanipulated to better fit the site. Optionally, the composite is thencaused to be set. The composite may be set by the addition of an agentsuch as a chemical agent, addition of energy such as UV light, oraddition of heat. In some embodiments, the composite is set by allowingthe implanted composite to cool to body temperature or by allowing asolvent or plasticizer to diffuse out from the composite.

The size of the particles may also dictate the delivery technique. Thedevice used to deliver the composite should have a sufficient diameterthat the particles do not clog the device. The particles may also betreated to reduce clogging, for example, by smoothing their surfaces,coating the particles, surface treating to improve their lubricity, orsimply reducing their size.

All of these factors may be easily optimized for a particular injectionsite. Theoretical discussions of the factors described above are foundin Bohner, et al., Biomaterials, (2003) 24:2721-2730, and Bohner, etal., (2005) 26:1553-1563, the contents of both of which are incorporatedherein by reference. The characteristics of various types of injectionsites (e.g., osteoporotic tissue, cancellous bone, cortical bone,substantially bone-free wound sites within bone) may be modeled withopen cell aluminum foam blocks (see Giannitsios, et al., European Cellsand Materials, Vol 10 Suppl 3 (2005), p. 54, the entire contents ofwhich are incorporated herein by reference). Such blocks may bereproducibly produced and thus are suitable for modeling various typesof bony tissue. For example, blocks may be produced with various poresizes and porosities and injected, e.g., first with polymers ofdifferent viscosities to identify an optimal viscosity range, and thenwith polymer mixtures within the viscosity range but having differentvolume fractions of particles. Alternatively or in addition, the porousblocks may also be produced to duplicate an individual patient's woundsite, which can be characterized using x-ray, MRI, and other imagingtechniques.

In some embodiments, the composite is heated above the glass transitiontemperature of the polymer component in preparation for injection orextrusion. As discussed above, where the glass transition temperature isgreater than body temperature, it should not be heated to temperature sogreat that either the tissue site or biological material in thecomposite is damaged. If the composite does not need to be held at anelevated temperature for a long period of time, a higher temperature maybe used without damaging biological materials.

As discussed herein, in some embodiments, the mixture includes amonomer, prepolymer, or telechelic polymer that is polymerized in situ.An initiator or catalyst may be injected into the tissue site as part ofthe composite or after the composite is injected. Alternatively or inaddition, the mixture is exposed to conditions that stimulatepolymerization after injection. In another embodiment, a lower molecularweight polymer is used in the composite and then cross-linked and/orfurther polymerized following implantation. Of course, if a polymer issufficiently viscous at body temperature, even if that is greater thanthe glass transition temperature, no pre- or post-injection processingof the mixture may be necessary.

After implantation, the composite typically stays at the site ofimplantation and is gradually resorbed by the body as bone forms in andaround it. The composite is typically engineered to provide themechanical strength necessary for the implantation site. The compositemay be resorbed after approximately 1 month to approximately 6 years.The resorption rate will depend on the polymer used in the composite,the site of implantation, the patient, disease condition, etc. Incertain embodiments, the composite lasts from approximately 1 month toapproximately 6 months. In other embodiments, the composite lasts fromapproximately 6 months to approximately 1 year. In other embodiments,the composite lasts from approximately 1-2 years. In other embodiments,the composite lasts from approximately 2-3 years. In other embodiments,the composite lasts from approximately 5 years.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1 50:50 Bone Particles and Polycaprolactone

A 50:50 mixture (by weight) of bovine bone fibers averaging 1-2 mm inlength and polycaprolactone was placed into a test tube. The compositewas heated in a plastic syringe in a microwave oven until thetemperature exceeded 60° C. The syringe was placed in a hand-drivenpress and the composite injected out. A similar composite was preparedwith 200-500 micron particles and exhibited improved extrusioncharacteristics.

A 50:50 mixture (by weight) of bovine bone fiber (300-800 micronssieved) and polycaprolactone were mixed into a uniform composite byheating the polymer in a microwave until melted. The composite was thenmixed by hand and the heating and melting repeated until the mixture wasuniform. A 65:35 mixture was formed in the same manner. A 50:50 mixtureof polycaprolactone and bone fibers, with 10% polyethylene glycol (PEG)(10,000 Da) was produced by first melting the PEG and then adding thebone particles. The polycaprolactone was then melted and the compositehand-mixed until uniform.

The 50:50/PEG composite was heated between two platens until moldableand fit into a cylindrical housing having a 3.3 mm ID×5 cm cannula onone end and a piston on the other. The assembled housing was submergedin a 70° C. water bath for about 10 minutes. The composite was forcedout of the cannula by driving the piston. The composite extruded at auniform rate from the cannula at about 0.5 cm/s. About 5 cm³ of thecomposite was extruded.

The composite was injected into a cortical defect. The composite moldeditself to the shape of the defect. Once the composite cooled (about 1minute), a rigid composite plug remained in the defect.

Example 2 Sawbone Trial

A composite of 60/40 (bovine bone fiber/polycaprolactone (PCL)) washeated to approximately 65° C. and packed into SAWBONES™ defects of thefollowing approximate shapes and sizes:

-   -   1. 3 cm diameter×1 cm depth defect.    -   2. A small defect 1 cm diameter×1 cm depth.    -   3. Lined the entire acetabular inner surface with polymer thinly        spread over the inner surface.    -   4. Many other Sawbones defects of various sizes.

Upon cooling and setting, the composite was either very difficult toremove or could not be removed from the defect by hand for all the abovecases.

For some of the above defects (#1 and #3), pilot holes were drilled andmetal screws with different thread profiles were placed into the PCLcomposite. The screws cut their own threads into the material. The PCLcomposite (when warm and moldable) was also placed around the threads ofvarious metal screws with different thread profiles. In both the tappedand moldable cases, using the protruding part of the screw as a“handle”, the composite/screw combination could not be removed from thedefect, and the screw could not be pulled from the PCL composite byhand.

Example 3 Excised Rabbit Femur Cylindrical Defect Trials

In another example, a 4.8 mm drill bit was used to create anapproximately 10 mm long defect in the distal part of a wet, excisedrabbit femur at room temperature. The defect was packed in one femur byheating 80/20 PCL/bovine bone particles to approximately 65° C., formingthe composite by hand into the rough shape of a cylinder with a diametersmaller than the defect, and then placing the cylinder in the defect,followed by immediately tamping the material into the defect to fill it.Excess material on the outside of the defect was removed (sheared off byhand) before the composite cooled. Upon cooling, the composite could notbe pulled out of the wet defect by hand.

In another case, using the same defect as described above (i.e., anapproximately 10 mm long defect in the distal part of a wet, excisedrabbit femur at room temperature), a composite of 50/50 PCL/bovine boneparticles was heated to approximately 65° C. and small portions of thecomposite were pinched off and packed into the defect with the aid of asmall cylindrical tamp. Approximately three small pieces of the warmcomposite were packed into the defect, one on top of the other, untilthe defect was filled. Upon cooling, the composite could not be pulledout of the wet defect by hand.

Water could be used to induce the composite to set more quickly.Irrigation with room temperature saline may achieve the same effect. Themoldable composite interdigitated in the crevices of the individual hosttrabeculae thereby anchoring the implant when it cooled to a rigid form.

Example 4 Moldable Bone/Polymer Composite

Three composites of bone fibers and poly(caprolactone) (PCL) wereprepared using the following percentages of bone fibers and polymer.

% Bone Fibers % PCL 50 50 65 35 80 20The percentages are by weight. A total of 1 g of each composite wasprepared.

The appropriate amount of poly(caprolactone) (inherent viscosity of 1.08dl/g) was weighed out and heated to approximately 100° C. forapproximately 5-10 minutes until the polymer softened. The appropriateamount of rabbit bone fibers, which had been sieved to between 0.85 mmand 0.30 mm, was then added to the melted polymer and mixed into thepolymer until the mixture was substantially homogenous. The compositewas then cooled to room temperature and packaged in sterile bags andsealed in foil pouches before sterilization by terminal gammairradiation. Five samples of each of the three composites were prepared.

The composites were graded according to how the heated composite feelsto the touch and how well it conforms when packing a small void. Eachcomposite was given a grade on a scale from 1 to 5 (1=poor handling,5=optimal handling). The grades represent the following handling of thecomposite.

Grade 1—Material is brittle and crumbly; material does not hold togetherwell (i.e., falls apart when handled); lack of cohesiveness between boneand polymer is extremely noticeable.

Grade 2—Material holds together somewhat but may be overly wet or sticky(e.g., sticks to the handler's gloves rather than defect); materialtakes a long time to set up or to soften; material often migrates fromthe defect site; and/or the material hardens to rapidly to manipulateinto a defect site (e.g., sets up in less than 1 minute, less than 1minute of working time).

Grade 3—Material is cohesive and pliable for at least 1-2 minutes ofworking time; material may stick lightly to the handler's gloves but iseasily removed; and/or material stays in the defect site with littletrouble or packs in easily.

Grade 4—Material is cohesive and pliable and has a working time of 2-4minutes; material does not stick to the handler's gloves; and/ormaterial is easily packing into a defect site.

Grade 5—Material is cohesive and pliable for 4-6 minutes; material doesnot stick to the handler's gloves and is easily packed into a defectsite.

The 50/50 bone/polymer composite was extremely easy to work with. Thecomposite could be manipulated for several minutes before setting up. Ona scale of 1 to 5 (5 being the best), this composite was rated a 4 forhandling and moldability.

The 65/35 bone/polymer composite was more difficult to prepare due tothe greater quantity of bone fibers that had to be worked into thepolymer. Handling of this composite was somewhat more difficult, and thecomposite setup more quickly. Handling for this composite was rated a 3.

The 8020 bone/polymer composite was even more difficult to prepare. Thesamples were crumbly with bone fibers falling out of the polymer.Handling for this composite was rated a 1.

Various mechanical properties of bone fiber/poly(caprolactone) (PCL)composites (70/30 bone/polymer and 50/50 bone/polymer) are given in thetable below entitled “Mechanical Testing Summary”.

Mechanical Testing Summary Compression Fatigue Yield 25 MPa Resid.Sample Stress (million Yld. Str. Molding Polymer (ratio) N (Mpa) Ncycles) (Mpa) Hydration Method PCL Fibers 10 21.5 + 0.7 4 1.11 + 0.1**22.99 14 day Comp. (70/30) M. PCL Fibers 2 25.6 + 0.0  1 day Hand(50/50) Mold **= some samples failed

Example 5 Moldable Bone/Polymer Composite

The following exemplary bone/polymer composites were prepared and testedfor their mechanical properties after hydration. The composites werealso rated on a scale of 1 to 5 (5 being the best) for their molding andhandling properties. The grading scale for the handling is describedabove in Example 4. The molding rating is simply the number of cyclesrequired on a microwave set at the “popcorn” setting to make itmoldable. Lower numbers are typically more desirable than highernumbers.

Average Hydration Compressive Average Compressive Molding HandlingIncubation Strength Modulus Strength Modulus Composite Rating RatingTime (hrs.) (MPa) (MPa) (MPa) (MPa) 80% poly-L-lactide-co-glycolide(82:18) 5 1 24 33.93 426.78 32.14 362.35 (Resomer 824) 35.72 968.73 20%PEG 37.5% poly-L-lactide-co-D,L-lactide (70:30) 1.5 2.5 168 3.38 89.963.42 87.04 (Resomer LR706) 3.33 92.88 30% PEG-8000 37.5% Bone fibers 40%poly-L-lactide-co-glycolide (75:25) 1.5 2.5 188 7.82 113.245 5.26 24.35(Purac) 10.38 202.14 30% PEG-8000 30% Bone fibers 38%PEGylated-poly-D,L-lactide (Lakeshore) 1.5 3.5 168 21.645 357.35 21.05386.45 24% PEG-8000 22.24 328.25 38% Bone fibers 50%poly-D,L-lactide-co-caprolactone (96/4) 1.5 4 168 10% PEG-8000 40% Bonefibers 50% poly-D,L-lactide-co-caprolactone (96/4) 0.75 4.5 24 30.761209.76 20% PEG-8000 30% Bone fibers 50% PEGylated poly-D,L-lactide(Lakeshore) 1 2 10% PEG-8000 40% Bone fibers 50%poly-L-lactide-co-glycolide (75:25) 1 3.5 24 30.39 672.64 (Purac) 30%PEG-8000 20% Bone fibers 46.7% poly-D,L-lactide-co-glycolide 3 4 24 41.21406.3 41.2 1406.3 20% PEG 33% Bone fibers 50%poly-D,L-lactide-co-glycolide 2 3 24 60.39 1814.94 60.39 1814.94 10% PEG40% Bone fibers 40% poly-D,L-lactide-co-glycolide 1 4 24 46.54 1635.0146.54 1635.01 20% PEG 40% Bone fibers 40% poly-D,L-lactide-co-glycolide1.5 3.5 24 65.05 1886.65 65.05 1886.65 10% PEG 50% Bone fibers 60%poly-D,L-lactide-co-glycolide 1 4 24 47.79 1502.37 47.79 1502.37 20% PEG20% Bone fibers 40% poly-D,L-lactide-co-glycolide 1 4 24 11.04 222.911.04 222.9 40% PEG 20% Bone fibers 50% poly-D,L-lactide-co-glycolide1.5 3 24 38.89 733.975 38.89 733.975 10% PEG 40% Bone fibers 40%poly-D,L-lactide-co-glycolide 1 4 24 27.64 666.125 27.64 666.125 20% PEG40% Bone fibers

EQUIVALENTS AND SCOPE

The foregoing has been a description of certain non-limiting preferredembodiments of the invention. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. Those of ordinary skill in the art will appreciate that variouschanges and modifications to this description may be made withoutdeparting from the spirit or scope of the present invention, as definedin the following claims.

In the claims articles such as “a”, “an”, and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the claims or from relevant portions of thedescription is introduced into another claim. For example, any claimthat is dependent on another claim can be modified to include one ormore limitations found in any other claim that is dependent on the samebase claim. Furthermore, where the claims recite a composition, it is tobe understood that methods of using the composition for any of thepurposes disclosed herein are included, and methods of making thecomposition according to any of the methods of making disclosed hereinor other methods known in the art are included, unless otherwiseindicated or unless it would be evident to one of ordinary skill in theart that a contradiction or inconsistency would arise. In addition, theinvention encompasses compositions made according to any of the methodsfor preparing compositions disclosed herein.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It is alsonoted that the term “comprising” is intended to be open and permits theinclusion of additional elements or steps. It should be understood that,in general, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, steps, etc.,certain embodiments of the invention or aspects of the inventionconsist, or consist essentially of, such elements, features, steps, etc.For purposes of simplicity those embodiments have not been specificallyset forth in haec verba herein. Thus for each embodiment of theinvention that comprises one or more elements, features, steps, etc.,the invention also provides embodiments that consist or consistessentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise. It is also to be understood that unlessotherwise indicated or otherwise evident from the context and/or theunderstanding of one of ordinary skill in the art, values expressed asranges can assume any subrange within the given range, wherein theendpoints of the subrange are expressed to the same degree of accuracyas the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment ofthe present invention may be explicitly excluded from any one or more ofthe claims. Any embodiment, element, feature, application, or aspect ofthe compositions and/or methods of the invention can be excluded fromany one or more claims. For purposes of brevity, all of the embodimentsin which one or more elements, features, purposes, or aspects isexcluded are not set forth explicitly herein.

What is claimed is:
 1. A composite osteoimplant comprising: a pluralityof particles comprising an inorganic material, a bone substitutematerial, a bone-derived material, or any combination thereof; and apolymer with which the plurality of particles has been combined; whereinthe composite is moldable or flowable; and wherein the composite becomesset upon exposure to suitable conditions for setting the composite, thesuitable conditions comprising cooling.
 2. The composite osteoimplant ofclaim 1, wherein the polymer has penetrated the pores, spaces, or voidsof the particles.
 3. The composite osteoimplant of claim 1, wherein thecomposite is moldable.
 4. The composite osteoimplant of claim 1, whereinthe composite can be shaped manually.
 5. The composite osteoimplant ofclaim 1, wherein the composite can be shaped using a surgicalinstrument.
 6. The composite osteoimplant of claim 1, wherein thecomposite can be shaped using a machine.
 7. The composite osteoimplantof claim 1, wherein the composite is injectable.
 8. The compositeosteoimplant of claim 7, whereby the composite is suitable for injectionthrough a 3 gauge or narrower needle.
 9. The composite osteoimplant ofclaim 7, whereby the composite is suitable for injection through a 5gauge or narrower needle.
 10. The composite osteoimplant of claim 7,whereby the composite is suitable for injection through a 7 gauge ornarrower needle.
 11. The composite osteoimplant of claim 7, whereby thecomposite is suitable for injection through a 10 gauge or narrowerneedle.
 12. The composite osteoimplant of claim 7, whereby the compositeis suitable for injection through a 12 gauge or narrower needle.
 13. Thecomposite osteoimplant of claim 1, wherein the suitable conditions forsetting the composite include changing the temperature of the composite,changing the osmotic pressure of the composite, exposing the compositeto electromagnetic radiation, cross-linking the composite, exposing thecomposite to a chemical agent, changing the water or solvent content ofthe composite, changing the content of a component of the composite, orchanging a diffusion gradient.
 14. The composite osteoimplant of claim1, wherein the suitable conditions for setting the composite includescooling the composite to body temperature of approximately 37° C.
 15. Acomposite osteoimplant comprising: a plurality of bone-derivedparticles; and a polymer with which the particles have been combined;wherein the composite has an initial phase and a set phase; wherein theset phase is more resistant to mechanical deformation relative to theinitial phase and the composite transitions to the set phase uponexposure to suitable conditions comprising cooling.
 16. A compositeosteoimplant comprising: a plurality of bone-derived particles; and apolymer with which the particles have been combined; wherein thecomposite has a first phase and a second phase; wherein the second phaseis more resistant to mechanical deformation relative to the first phaseand the composite transitions to the second phase upon exposure tosuitable conditions comprising cooling.
 17. The composite osteoimplantof claim 16, wherein the composite can be converted reversibly betweenthe first and second phase.
 18. The composite osteoimplant of claim 16,wherein conversion from the first to second phase is irreversible.
 19. Acomposite osteoimplant comprising: a plurality of bone-derivedparticles; and a polymer with which the particles have been combined;wherein the composite has a first phase and a second phase; wherein thefirst phase is more moldable, flowable, or injectable relative to thesecond phase and the composite transitions to the second phase uponexposure to suitable conditions comprising cooling.